Prostate-specific membrane antigen and uses thereof

ABSTRACT

This invention provides an isolated nucleic acid molecule encoding an alternatively spliced human prostate-specific membrane antigen. This invention provides an isolated nucleic acid comprising a promoter sequence normally associated with the transcription of a gene encoding a human prostate-specific membrane antigen. This invention provides an isolated polypeptide having the biological activity of an alternatively spliced prostate-specific membrane antigen. 
     This invention provides a method of detecting a nucleic acid encoding an alternatively spliced human prostate-specific membrane antigen and a method of detecting a prostate tumor cell in a subject. 
     Lastly, this invention provides a pharmaceutical composition comprising a compound in a therapeutically effective amount and a pharmaceutically acceptable carrier and a method of making prostate cells susceptible to a cytotoxic chemotherapeutic agent.

This application is a continuation-in-part of International ApplicationPCT/US96/02424, filed Jul. 19, 1996, which is a continuation-in-partapplication of U.S. application Ser. No. 08/394,152, filed Feb. 24,1995, the contents of which are hereby incorporated by reference.

This invention disclosed herein was made in part with Government supportunder Grants Nos. DK47650 and CA58192, CA-39203, CA-29502, CA-08748-29from the National Institute of Health, U.S. Department of Health andHuman Services. Accordingly, the U.S. Government has certain rights inthis invention.

BACKGROUND OF THE INVENTION

Throughout this application various references are referred to withinparentheses. Disclosures of these publications in their entireties arehereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains. Fullbibliographic citation for these references may be found at the end ofeach set of Examples in the Experimental Details section.

Prostate cancer is among the most significant medical problems in theUnited States, as the disease is now the most common malignancydiagnosed in American males. In 1992 there were over 132,000 new casesof prostate cancer detected with over 36,000 deaths attributable to thedisease, representing a 17.3% increase over 4 years (8). Five yearsurvival rates for patients with prostate cancer range from 88% forthose with localized disease to 29% for those with metastatic disease.The rapid increase in the number of cases appears to result in part froman increase in disease awareness as well as the widespread use ofclinical markers such as the secreted proteins prostate-specific antigen(PSA) and prostatic acid phosphatase (PAP) (7).

The prostate gland is a site of significant pathology affected byconditions such as benign growth (BPH), neoplasia (prostatic cancer) andinfection (prostatitis). Prostate cancer represents the second leadingcause of death from cancer in man (7). However prostatic cancer is theleading site for cancer development in men. The difference between thesetwo facts relates to prostatic cancer occurring with increasingfrequency as men age, especially in the ages beyond 60 at a time whendeath from other factors often intervenes. Also, the spectrum ofbiologic aggressiveness of prostatic cancer is great, so that in somemen following detection the tumor remains a latent histologic tumor anddoes not become clinically significant, whereas in other it progressesrapidly, metastasizes and kills the man in a relatively short 2-5 yearperiod (7 and 59).

In prostate cancer cells, two specific proteins that are made in veryhigh concentrations are prostatic acid phosphatase (PAP) and prostatespecific antigen (PSA) (21, 47, and 65). These proteins have beencharacterized and have been used to follow response to therapy. With thedevelopment of cancer, the normal architecture of the gland becomesaltered, including loss of the normal duct structure for the removal ofsecretions and thus the secretions reach the serum. Indeed measurementof serum PSA is suggested as a potential screening method for prostaticcancer. Indeed, the relative amount of PSA and/or PAP in the cancerreduces as compared to normal or benign tissue. PAP was one of theearliest serum markers for detecting metastatic spread (47). PAPhydrolyses tyrosine phosphate and has a broad substrate specificity.Tyrosine phosphorylation is often increased with oncogenictransformation. It has been hypothesized that during neoplastictransformation there is less phosphatase activity available toinactivate proteins that are activated by phosphorylation on tyrosineresidues. In some instances, insertion of phosphatases that havetyrosine phosphatase activity has reversed the malignant phenotype.

PSA is a protease and it is not readily appreciated how loss of itsactivity correlates with cancer development (21, and 65). Theproteolytic activity of PSA is inhibited by zinc. Zinc concentrationsare high in the normal prostate and reduced in prostatic cancer.Possibly the loss of zinc allows for increased proteolytic activity byPSA. As proteases are involved in metastasis and some proteasesstimulate mitotic activity, the potentially increased activity of PSAcould be hypothesized to play a role in the tumors metastases and spread(39).

Both PSA and PAP are found in prostatic secretions. Both appear to bedependent on the presence of androgens for their production and aresubstantially reduced following androgen deprivation.

Prostate-specific membrane antigen (PSM) which appears to be localizedto the prostatic membrane has been identified. This antigen wasidentified as the result of generating monoclonal antibodies to aprostatic cancer cell, LNCaP (22).

Dr. Horoszewicz established a cell line designated LNCaP from the lymphnode of a hormone refractory, heavily pretreated patient (23). This linewas found to have an aneuploid human male karyotype. It maintainedprostatic differentiation functionality in that it produced both PSA andPAP. It possessed an androgen receptor of high affinity and specificity.Mice were immunized with LNCaP cells and hybridomas were derived fromsensitized animals. A monoclonal antibody was derived and was designated7E11-C5 (22). The antibody staining was consistent with a membranelocation and isolated fractions of LNCaP cell membranes exhibited astrongly positive reaction with immunoblotting and ELISA techniques.This antibody did not inhibit or enhance the growth of LNCaP cells invitro or in vivo. The antibody to this antigen was remarkably specificto prostatic epithelial cells, as no reactivity was observed in anyother component. Immunohistochemical staining of cancerous epithelialcells was more intense than that of normal or benign epithelial cells.

Dr. Horoszewicz also reported detection of immunoreactive material using7E11-C5 in serum of prostatic cancer patients (22). The immunoreactivitywas detectable in nearly 60% of patients with stage D-2 disease and in aslightly lower percentage of patients with earlier stage disease, butthe numbers of patients in the latter group are small. Patients withbenign prostatic hyperplasia (BPH) were negative. Patients with noapparent disease were negative, but 50-60% of patients in remission yetwith active stable disease or with progression demonstrated positiveserum reactivity. Patients with non prostatic tumors did not showimmunoreactivity with 7E11-C5.

The 7E11-C5 monoclonal antibody is currently in clinical trials. Thealdehyde groups of the antibody were oxidized and the linker-chelatorglycol-tyrosyl-(n,ε-diethylenetriamine-pentacetic acid)-lysine(GYK-DTPA) was coupled to the reactive aldehydes of the heavy chain. Theresulting antibody was designated CYT-356. Immunohistochemical stainingpatterns were similar except that the CYT-356 modified antibody stainedskeletal muscle. The comparison of CYT-356 with 7E11-C5 monoclonalantibody suggested both had binding to type 2 muscle fibers. The reasonfor the discrepancy with the earlier study, which reported skeletalmuscle to be negative, was suggested to be due to differences in tissuefixation techniques. Still, the most intense and definite reaction wasobserved with prostatic epithelial cells, especially cancerous cells.Reactivity with mouse skeletal muscle was detected withimmunohistochemistry but not in imaging studies. The Indium¹¹¹-labeledantibody localized to LNCaP tumors grown in nude mice with an uptake ofnearly 30% of the injected dose per gram tumor at four days. In-vivo, noselective retention of the antibody was observed in antigen negativetumors such as PC-3 and DU-145, or by skeletal muscle. Very little wasknown about the PSM antigen. An effort at purification andcharacterization has been described at meetings by Dr. George Wright andcolleagues (14 and 64).

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C:

-   -   Immunohistochemical detection of PSM antigen expression in        prostate cell lines. Top panel reveals uniformly high level of        expression in LNCaP cells; middle panel and lower panel are        DU-145 and PC-3 cells respectively, both negative.

FIG. 2: Autoradiogram of protein gel revealing products of PSM coupledin-vitro transcription/translation. Non-glycosylated PSM polypeptide isseen at 84 kDa (lane 1) and PSM glycoprotein synthesized following theaddition of microsomes is seen at 100 kDa (lane 2).

Western Blot analysis detecting PSM expression in transfected non-PSMexpressing PC-3 cells. 100 kDa PSM glycoprotein species is clearly seenin LNCaP membranes (lane 1), LNCaP crude lysate (lane 2), andPSM-transfected PC-3 cells (lane 4), but is undetectable in native PC-3cells (lane 3).

FIG. 4: Autoradiogram of ribonuclease protection gel assaying for PSMmRNA expression in normal human tissues. Radiolabeled 1 kb DNA ladder(Gibco-BRL) is shown in lane 1. Undigested probe is 400 nucleotides(lane 2), expected protected PSM band is 350 nucleotides, and tRNAcontrol is shown (lane 3). A strong signal is seen in human prostate(lane 11), with very faint, but detectable signals seen in human brain(lane 4) and human salivary gland (lane 12). No signal was detected inlane 5 kidney, lane 6 liver, lane 7 lung, lane 8 mammary gland, lane 9pancreas, lane 10 placenta, lane 13 skeletal muscle, lane 14 spleen, andlane 15 testes.

FIG. 5: Autoradiogram of ribonuclease protection gel assaying for PSMmRNA expression in LNCaP tumors grown in nude mice, and in humanprostatic tissues. ³²P-labeled 1 kb DNA ladder is shown in lane 1. 298nucleotide undigested probe is shown (lane 2), and tRNA control is shown(lane 3). PSM mRNA expression is clearly detectable in LNCaP cells (lane4), orthotopically grown LNCaP tumors in nude mice with and withoutmatrigel (lanes 5 and 6), and subcutaneously implanted and grown LNCaPtumors in nude mice (lane 7). PSM mRNA expression is also seen in normalhuman prostate (lane 8), and in a moderately differentiated humanprostatic adenocarcinoma (lane 10). Very faint expression is seen in asample of human prostate tissue with benign hyperplasia (lane 9).

FIG. 6: Ribonuclease protection assay for PSM expression in LNCaP cellstreated with physiologic doses of various steroids for 24 hours.³²P-labeled DNA ladder is shown in lane 1. 298 nucleotide undigestedprobe is shown (lane 2), and tRNA control is shown (lane 3). PSM mRNAexpression is highest in untreated LNCaP cells in charcoal-strippedmedia (lane 4). Applicant see significantly diminished PSM expression inLNCaP cells treated with DHT (lane 5), Testosterone (lane 6), Estradiol(line 7), and Progesterone (lane 8), with little response toDexamethasone (lane 9).

FIG. 7: Data illustrating results of PSM DNA and RNA presence intransfect Dunning cell lines employing Southern and Northern blottingtechniques

FIGS. 8A-8B:

-   -   Figure A indicates the power of cytokine transfected cells to        teach unmodified cells. Administration was directed to the        parental flank or prostate cells. The results indicate the        microenvironment considerations.    -   Figure B indicates actual potency at a particular site. The        tumor was implanted in prostate cells and treated with immune        cells at two different sites.

FIGS. 9A-9B:

-   -   Relates potency of cytokines in inhibiting growth of primary        tumors. Animals administered un-modified parental tumor cells        and administered as a vaccine transfected cells. Following        prostatectomy of rodent tumor results in survival increase.

FIG. 10: PCR amplification with nested primers improved the level ofdetection of prostatic cells from approximately one prostatic cell per10,000 MCF-7 cells to better than one cell per million MCF-7 cells,using PSA.

FIG. 11: PCR amplification with nested primers improved the level ofdetection of prostatic cells from approximately one prostatic cell per10,000 MCF-7 cells to better than one cell per million MCF-7 cells,using PSM-derived primers.

FIG. 12: A representative ethidium stained gel photograph for PSM-PCR.Samples run in lane A represent PCR products generated from the outerprimers and samples in lanes labeled B are products of inner primerpairs.

FIG. 13: PSM Southern blot autoradiograph. The sensitivity of theSouthern blot analysis exceeded that of ethidium staining, as can beseen in several samples where the outer product is not visible, but isdetectable by Southern blotting.

FIG. 14: Characteristics of the 16 patients analyzed with respect totheir clinical stage, treatment, serum PSA and PAP values, and resultsof assay.

FIGS. 15A-15D:

-   -   DNA sequence containing promoter elements from nucleotide −1 to        nucleotide −3017. −1 is upstream of start site of PSM.

FIG. 16: Potential binding sites on the PSM promoter fragment.

FIG. 17: Promoter activity of PSM up-stream fragment/CAT gene chimera.

FIG. 18: Comparison between PSM and PSM′ cDNA. Sequence of the 5′ end ofPSM cDNA (32) is shown. Underlined region (beginning at nucleotide 115and continuing to nucleotide 380) denotes nucleotides which are absentin PSM′ cDNA but present in PSM cDNA. Boxed region represents theputative transmembrane domain of PSM antigen. * Asterisk denotes theputative translation initiation site for PSM′.

FIG. 19: Graphical representation of PSM and PSM′ cDNA sequences andantisense PSM RNA probe (b). PSM cDNA sequence with complete codingregion (32). (a) PSM′ cDNA sequence from this study. (c) Cross hatchedand open boxes denote sequences identity in PSM and PSM′. Hatched boxindicates sequence absent from PSM′. Regions of cDNA sequencecomplementary to the antisense probe are indicated by dashed linesbetween the sequences.

FIG. 20: RNase protection assay with PSM specific probe in primaryprostatic tissues. Total cellular RNA was isolated from human prostaticsamples: normal prostate, BPH, and CaP. PSM and PSM′ spliced variantsare indicated with arrows at right. The left lane is a DNA ladder.Samples from different patients are classified as: lanes 3-6, CaP,carcinoma of prostate; BPH, benign prostatic hypertrophy, lanes 7-9;normal, normal prostatic tissue, lanes 10-12. Autoradiograph was exposedfor longer period to read lanes 5 and 9.

FIG. 21: Tumor Index, a quantification of the expression of PSM andPSM′. Expression of PSM and PSM′ was quantified by densitometry andexpressed as a ratio of PSM/PSM′ on the Y-axis. Three samples each werequantitated for primary CaP, BPH and normal prostate tissues. Twosamples were quantitated for LNCaP. Normal, normal prostate tissue.

FIG. 22: Characterization of PSM membrane bound and PSM′ in the cytosol.

FIG. 23: Photograph of ethidium bromide stained gel depictingrepresentative negative and positive controls used in the study. Samples1-5 were from, respectively: male with prostatis, a healthy femalevolunteer, a male with BPH, a control 1:1,000,000 dilution of LNCaPcells, and a patient with renal cell carcinoma. Below each reaction isthe corresponding control reaction performed with beta-2-microglobulinprimers to assure RNA integrity. No PCR products were detected for anyof these negative controls.

FIG. 24: Photograph of gel displaying representative positive PCRresults using PSM primers in selected patients with either localized ordisseminated prostate cancer. Sample 1-5 were from respectively: apatient with clinically localized stage T1_(c) disease, a radicalprostatectomy patient with organ confined disease and a negative serumPSA, a radical prostatectomy patient with locally advanced disease and anegative serum PSA, a patient with treated stage D2 disease, and apatient with treated hormone refractory disease.

FIG. 25: Chromosomal location of PSM based on in-situ hybridization withcDNA and with genomic cosmids.

FIG. 26: Human monochromosomal somatic cell hybrid blot showing thatchromosome 11 contained the PSM genetic sequence by Southern analysis.DNA panel digested with PstI restriction enzyme and probed with PSMcDNA. Lanes M and H refer to mouse and hamster DNAs. The numberscorrespond to the human chromosomal DNA in that hybrid.

FIG. 27: Ribonuclease protection assay using PSM radiolabeled RNA proberevels an abundant PSM mRNA expression in AT6.1-11 clone 1, but not inAT6.1-11 clone 2, thereby mapping PSM to 11p11.2-13 region.

FIG. 28: Tissue specific expression of PSM RNA by Northern blotting andRNAse protection assay.

FIG. 29: Mapping of the PSM gene to the 11p11.2-p13 region of humanchromosome 11 by southern blotting and in-situ hybridization.

FIG. 30: Schematic of potential response elements.

FIG. 31: Schematic depiction of metastatic prostate cell transfectedwith promoter for PSM which is driving expression of prodrug activatingenzyme cytosine deaminase. This allows for prostate specific expressionand tumor localized conversion of non-toxic 5 fluorocytosine to 5fluorouracil.

FIG. 32A-32C:

-   -   Nucleic acid of PSM genomic DNA is read 5 prime away from the        transcription start site: number on the sequences indicates        nucleotide upstream from the start site. Therefore, nucleotide        #121 is actually −121 using conventional numbering system.

FIG. 33: Representation of NAAG 1, acividin, azotomycin, and6-diazo-5-oxo-norleucine, DON.

FIG. 34: Representation of N-acetylaspartylglutamate (NAAG), PALA,PALAGLU, phosphonate antagonist of glutamate receptor and phosphonatesof PALAGLU and NAAG.

FIG. 35: Synthesis of N-acetylaspartylglutamate, NAAG 1.

FIG. 36: Synthesis of N-phosphonoacetylaspartyl-L-glutamate.

FIG. 37: Synthesis of 5-diethylphosphonon-2 amino benzylvalerateintermediate.

FIG. 38: Synthesis of analog 4 and 5.

FIG. 39: Representation of DON, analogs 17-20.

FIG. 40: Substrates for targeted drug delivery, analog 21 and 22.

FIG. 41: Dynemycin A and its mode of action.

FIG. 42: Synthesis of analog 28.

FIG. 43: Synthesis for intermediate analog 28.

FIG. 44: Attachment points for PALA.

FIG. 45: Mode of action for substrate 21.

FIGS. 46A-46D:

-   -   Intron 1F: Forward Sequence.

FIGS. 47A-47E:

-   -   Intron 1R: Reverse Sequence

FIGS. 48A-48C:

-   -   Intron 2F: Forward Sequence

FIGS. 49A-49C:

-   -   Intron 2R: Reverse Sequence

FIGS. 50A-508:

-   -   Intron 3F: Forward Sequence

FIGS. 51A-51B:

-   -   Intron 3R: Reverse Sequence

FIGS. 52A-52C:

-   -   Intron 4F: Forward Sequence

FIGS. 53A-53E:

-   -   Intron 4RF: Reverse Sequence

FIG. 54: PSM genomic organization of the exon and 19 intron junctionsequences. The exon/intron junctions are as follows:

-   -   1. Exon/intron 1 at by 389-390;    -   2. Exon/intron 2 at by 490-491;    -   3. Exon/intron 3 at by 681-682;    -   4. Exon/intron 4 at by 784-785;    -   5. Exon/intron 5 at by 911-912;    -   6. Exon/intron 6 at by 1096-1097;    -   7. Exon/intron 7 at by 1190-1191;    -   8. Exon/intron 8 at by 1289-1290;    -   9. Exon/intron 9 at by 1375-1376;    -   10. Exon/intron 10 at by 1496-1497;    -   11. Exon/intron 11 at by 1579-1580;    -   12. Exon/intron 12 at by 1643-1644;    -   13. Exon/intron 13 at by 1710-1711;    -   14. Exon/intron 14 at by 1803-1804;    -   15. Exon/intron 15 at by 1894-1895;    -   16. Exon/intron 16 at by 2158-2159;    -   17. Exon/intron 17 at by 2240-2241;    -   18. Exon/intron 18 at by 2334-2335;    -   19. Exon/intron 19 at by 2644-2645.

FIGS. 55A-55J:

-   -   Alternatively spliced PSM (PSM′)nucleic acid sequence and amino        acid sequence.

FIG. 56: PSM pteroyl (folate) hydrolase activity in LNCaP membranepreparation. Time course of MTXglu₃ hydrolysis (-▪-) and concurrentformation of MTXglu₂ (- -), MTXglu₁ (-▴-), and MTX (- -), respectively.Membrane fractions were prepared as described in Methods. Reactionvolume was 100 μL containing 50 mM acetate/Triton buffer pH 4.5, 50 μMMTXglu₃, 10 μg/mL protein. Values are x±S.D. from three separate LNCaPmembrane preparations.

FIG. 57: PSM pteroyl (folate) hydrolase activity of immunoprecipitatedPSM antigen. Diagram shows typical capillary electrophoretic separationpatterns of MTXglu_((n)) derivatives at 0, 30, 60 and 240 minutereaction times. Elution intervals for MTXglu₃, MTXglu₂, MTXglu₁, and MTXare 4.25, 3.95, 3.55, and 3.06 min, respectively. Total volume ofreaction mixture was 100 uL containing 50 uM MTXglu₃.

FIG. 58: Effects of pH on gamma-glutamyl hydrolase (PSM hydrolase)activity in LNCaP, PC-3 PSM-transfected (PC-3(+)) and PSMnon-transfected (PC-3(−)) cells. Enzymic activity is reported as μMMTXglu₂ formed/mg protein. Each value represents the mean of 3 reactionscontaining 50-60 μg/mL protein. The following buffers were used in 50 mMconcentrations spanning a pH range of 2 to 10: glycine-HCl, pH 2.2 to3.6; acetate, pH 3.6 to 5.6; 2-(N-morpholino)ethanesulfonic acid (MES),pH 5.6 to 6.8; Tris(hydroxymethyl)aminomethane (TRIS), pH 7 to 8.5; andglycine-NaOH, pH 8.6 to 10.0.

FIG. 59: Comparison of pteroyl hydrolase activity in membranes isolatedfrom LNCaP, PC-3, TSU-Pr1, and Duke-145 adenocarcinoma cell lines.Membranes were isolated as described in Methods. Each value representsthe mean of triplicate reactions normalized to 1 mg/mL protein.

FIG. 60A-60C:

-   -   Immunohistochemical analysis of LNCaP and PC-3 PSM-transfected        and PSM-non-transfected cells. A 2.65 kb PSM cDNA containing a        hygromycin selection vector was cloned into non PSM-antigen        expressing PC-3 cells and maintained in regular media        supplemented with hygromycin B. As a control, PC-3 cells were        also transfected with the pREP7 vector alone (PC-3 PSM        non-transfected cells). Cells were permeabilized in        acetone/methanol (1:1 v/v) mixture, blocked with 5% bovine serum        albumin/Tris buffered saline (TBS) and the 7E11-C5 monoclonal        PSM antibody was added to cells. A secondary anti-mouse IgG₁        antibody conjugated with alkaline phosphatase was added and        PSM-positive cell staining performed with        bromochloroindolylphenol phosphate. Panel A demonstrates intense        immunoreactivity associated with LNCaP cells using the        monoclonal PSM antibody; In panel B, comparable staining occurs        in PC-3 cells transfected with PSM expression vector. Panel C        illustrates PC-3 cells expressing pREP7 hygromycin vector alone.

FIG. 61: Comparison of pteroyl (folate) hydrolase activity in membranesisolated from PSM expressing PC-3 cells and PC-3 cells expressing pREP7hygromycin vector alone. Membranes were isolated as described inMethods. Each value represents the mean of triplicate reactionsnormalized to 1 mg/mL protein.

FIG. 62: Representation of N-acetylaspartylglutamate (NAAG), folic acid,folate-gamma-polyglutamate, methotrexate,methotrexate-gamma-polyglutamate, methotrexate-alpha-monoglutamate,methotrexate-gamma-diglutamate, methotrexate-gamma-triglutamate,methotrexate-gamma-tetraglutamate.

FIG. 63A-63B:

-   -   Solid phase synthesis of methotrexate alpha-polyglutamatae        analogs.

FIG. 64: Sequence analysis of microsatellite instability in PSM gene.

FIG. 65: PSM genomic organization.

FIG. 66: Location of microsatellite in PSM gene

SUMMARY OF THE INVENTION

This invention provides an isolated nucleic acid molecule encoding analternatively spliced human prostate-specific membrane antigen. Thisinvention provides an isolated nucleic acid comprising a promotersequence normally associated with the transcription of a gene encoding ahuman prostate-specific membrane antigen. This invention provides anisolated polypeptide having the biological activity of an alternativelyspliced prostate-specific membrane antigen.

This invention provides a method of detecting a nucleic acid encoding analternatively spliced human prostate-specific membrane antigen and amethod of detecting a prostate tumor cell in a subject.

Lastly, this invention provides a pharmaceutical composition comprisinga compound in a therapeutically effective amount and a pharmaceuticallyacceptable carrier and a method of making prostate cells susceptible toa cytotoxic agent.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides an isolated nucleic acid encoding analternatively spliced human prostate-specific membrane (PSM′) antigen.As defined herein “nucleic acid encoding an alternatively splicedprostate-specific membrane (PSM′) antigen” means nucleic acid encoding aprostate-specific membrane antigen which contains a deletion in the DNAsequence encoding prostate specific membrane antigen between nucleotide115 and 380. In one embodiment the isolated nucleic acid encodes thealternatively spliced human prostate-specific membrane antigen as setforth in FIG. 55.

This invention further provides an isolated mammalian genomic DNAmolecule which encodes an alternatively spliced prostate-specificmembrane antigen. This invention further provides an isolated mammalianDNA molecule of an isolated mammalian nucleic acid molecule encoding analternatively spliced prostate-specific membrane antigen. This inventionalso provides an isolated mammalian cDNA molecule encoding a mammalianalternatively spliced prostate-specific membrane antigen. This inventionprovides an isolated mammalian RNA molecule encoding a mammalianalternatively spliced prostate-specific membrane antigen.

This invention also encompasses DNAs and cDNAs which encode amino acidsequences which differ from those of PSM′ antigen, but which should notproduce phenotypic changes. Alternatively, this invention alsoencompasses DNAs and cDNAs which hybridize to the DNA and cDNA of thesubject invention. Hybridization methods are well known to those ofskill in the art.

This invention also provides a nucleic acid molecule of at least 15nucleotides capable of specifically hybridizing with a sequence of anucleic acid molecule encoding the prostate-specific membrane antigen.This molecule may either be a DNA or RNA molecule.

This invention provides a nucleic acid sequence of at least 15nucleotides capable of specifically hybridizing to a sequence within aDNA sequence encoding prostate specific membrane antigen located betweennucleotide 115 and nucleotide 380.

The nucleic acid molecule capable of specifically hybridizing with asequence of a nucleic acid molecule encoding the prostate-specificmembrane antigen can be used as a probe. Nucleic acid probe technologyis well known to those skilled in the art who will readily appreciatethat such probes may vary greatly in length and may be labeled with adetectable label, such as a radioisotope or fluorescent dye, tofacilitate detection of the probe. DNA probe molecules may be producedby insertion of a DNA molecule which encodes PSM antigen into suitablevectors, such as plasmids or bacteriophages, followed by transforminginto suitable bacterial host cells, replication in the transformedbacterial host cells and harvesting of the DNA probes, using methodswell known in the art. Alternatively, probes may be generated chemicallyfrom DNA synthesizers.

RNA probes may be generated by inserting the PSM antigen moleculedownstream of a bacteriophage promoter such as T3, T7 or SP6. Largeamounts of RNA probe may be produced by incubating the labelednucleotides with the linearized PSM antigen fragment where it containsan upstream promoter in the presence of the appropriate RNA polymerase.

For example, high stringent hybridization conditions are selected atabout 5° C. lower than the thermal melting point (Tm) for the specificsequence at a defined ionic strength and pH. The Tm is the temperature(under defined ionic strength and pH) at which 50% of the targetsequence hybridizes to a perfectly matched probe. Typically, stringentconditions will be those in which the salt concentration is at leastabout 0.02 molar at pH 7 and the temperature is at least about 60° C. Asother factors may significantly affect the stringency of hybridization,including, among others, base composition and size of the complementarystrands, the presence of organic solvents, ie. salt or formamideconcentration, and the extent of base mismatching, the combination ofparameters is more important than the absolute measure of any one. ForExample high stringency may be attained for example by overnighthybridization at about 68° C. in a 6×SSC solution, washing at roomtemperature with 6×SSC solution, followed by washing at about 68° C. ina 6×SSC in a 0.6×SSX solution.

Hybridization with moderate stringency may be attained for exampleby: 1) filter pre-hybridizing and hybridizing with a solution of 3×sodium chloride, sodium citrate (SSC), 50% formamide, 0.1M Tris bufferat Ph 7.5, 5×Denhardt's solution; 2.) pre-hybridization at 37° C. for 4hours; 3) hybridization at 37° C. with amount of labelled probe equal to3,000,000 cpm total for 16 hours; 4) wash in 2×SSC and 0.1% SDSsolution; 5) wash 4× for 1 minute each at room temperature at 4× at 60°C. for 30 minutes each; and 6) dry and expose to film.

The DNA molecules described and claimed herein are useful for theinformation which they provide concerning the amino acid sequence of thepolypeptide and as products for the large scale synthesis of thepolypeptide by a variety of recombinant techniques. The molecule isuseful for generating new cloning and expression vectors, transformedand transfected prokaryotic and eukaryotic host cells, and new anduseful methods for cultured growth of such host cells capable ofexpression of the polypeptide and related products.

Moreover, the isolated mammalian nucleic acid molecules encoding amammalian prostate-specific membrane antigen and the alternativelyspliced PSM′ are useful for the development of probes to study thetumorigenesis of prostate cancer.

The nucleic acid molecules synthesized above may be used to detectexpression of a PSM′ antigen by detecting the presence of mRNA codingfor the PSM antigen. Total mRNA from the cell may be isolated by manyprocedures well known to a person of ordinary skill in the art. Thehybridizing conditions of the labelled nucleic acid molecules may bedetermined by routine experimentation well known in the art. Thepresence of mRNA hybridized to the probe may be determined by gelelectrophoresis or other methods known in the art. By measuring theamount of the hybrid made, the expression of the PSM and PSM′ antigen bythe cell can be determined. The labeling may be radioactive. For anexample, one or more radioactive nucleotides can be incorporated in thenucleic acid when it is made.

In one embodiment of this invention, nucleic acids are extracted byprecipitation from lysed cells and the mRNA is isolated from the extractusing an oligo-dT column which binds the poly-A tails of the mRNAmolecules. The mRNA is then exposed to radioactively labelled probe on anitrocellulose membrane, and the probe hybridizes to and thereby labelscomplementary mRNA sequences. Binding may be detected by luminescenceautoradiography or scintillation counting. However, other methods forperforming these steps are well known to those skilled in the art, andthe discussion above is merely an example.

The probes are also useful for in-situ hybridization or in order tolocate tissues which express this gene, or for other hybridizationassays for the presence of this gene or its mRNA in various biologicaltissues. The in-situ hybridization using a labelled nucleic acidmolecule is well known in the art. Essentially, tissue sections areincubated with the labelled nucleic acid molecule to allow thehybridization to occur. The molecule will carry a marker for thedetection because it is “labelled”, the amount of the hybrid will bedetermined based on the detection of the amount of the marker and sowill the expression of PSM antigen.

This invention further provides isolated PSM′ antigen nucleic acidmolecule operatively linked to a promoter of RNA transcription. Theisolated PSM′ antigen sequence can be linked to vector systems. Variousvectors including plasmid vectors, cosmid vectors, bacteriophage vectorsand other viruses are well known to ordinary skilled practitioners. Thisinvention further provides a vector which comprises the isolated nucleicacid molecule encoding for the PSM′ antigen.

As an example to obtain these vectors, insert and vector DNA can both beexposed to a restriction enzyme to create complementary ends on bothmolecules which base pair with each other and are then ligated togetherwith DNA ligase. Alternatively, linkers can be ligated to the insert DNAwhich correspond to a restriction site in the vector DNA, which is thendigested with the restriction enzyme which cuts at that site. Othermeans are also available and known to an ordinary skilled practitioner.

Plasmid, p55A-PSM, was deposited on Aug. 14, 1992 with the American TypeCulture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852,U.S.A. under the provisions of the Budapest Treaty for the InternationalRecognition of the Deposit of Microorganism for the Purposes of PatentProcedure. Plasmid, p55A-PSM, was accorded ATCC Accession Number 75294.

This invention further provides a host vector system for the productionof a polypeptide having the biological activity of the alternativelyspiced prostate-specific membrane antigen. These vectors may betransformed into a suitable host cell to form a host cell vector systemfor the production of a polypeptide having the biological activity ofPSM′ antigen.

Regulatory elements required for expression include promoter sequencesto bind RNA polymerase and transcription initiation sequences forribosome binding. For example, a bacterial expression vector includes apromoter such as the lac promoter and for transcription initiation theShine-Dalgarno sequence and the start codon AUG. Similarly, a eukaryoticexpression vector includes a heterologous or homologous promoter for RNApolymerase II, a downstream polyadenylation signal, the start codon AUG,and a termination codon for detachment of the ribosome. Such vectors maybe obtained commercially or assembled from the sequences described bymethods well known in the art, for example the methods described abovefor constructing vectors in general. Expression vectors are useful toproduce cells that express the PSM antigen.

This invention further provides an isolated DNA or cDNA moleculedescribed hereinabove wherein the host cell is selected from the groupconsisting of bacterial cells (such as E. coli), yeast cells, fungalcells, insect cells and animal cells. Suitable animal cells include, butare not limited to Vero cells, HeLa cells, Cos cells, CV1 cells andvarious primary mammalian cells.

This invention provides an isolated polypeptide having the biologicalactivity of an alternatively spliced prostate-specific membrane antigen.

This invention further provides a method of producing a polypeptidehaving the biological activity of the prostate-specific membrane antigenwhich comprising growing host cells of a vector system containing thePSM′ antigen sequence under suitable conditions permitting production ofthe polypeptide and recovering the polypeptide so produced.

This invention provides a mammalian cell comprising a DNA moleculeencoding a mammalian PSM′ antigen, such as a mammalian cell comprising aplasmid adapted for expression in a mammalian cell, which comprises aDNA molecule encoding a mammalian PSM′ antigen and the regulatoryelements necessary for expression of the DNA in the mammalian cell solocated relative to the DNA encoding the mammalian PSM′ antigen as topermit expression thereof.

Numerous mammalian cells may be used as hosts, including, but notlimited to, the mouse fibroblast cell NIH3T3, CHO cells, HeLa cells,Ltk⁻ cells, Cos cells, etc. Expression plasmids such as that describedsupra may be used to transfect mammalian cells by methods well known inthe art such as calcium phosphate precipitation, electroporation or DNAencoding the mammalian PSM antigen may be otherwise introduced intomammalian cells, e.g., by microinjection, to obtain mammalian cellswhich comprise DNA, e.g., cDNA or a plasmid, encoding a mammalian PSMantigen.

This invention further provides ligands bound to the mammalian PSM′antigen.

This invention also provides a therapeutic agent comprising a ligandidentified by the above-described method and a cytotoxic agentconjugated thereto. The cytotoxic agent may either be a radioisotope ora toxin. Examples of radioisotopes or toxins are well known to one ofordinary skill in the art.

This invention also provides a method of imaging prostate cancer inhuman patients which comprises administering to the patients at leastone ligand identified by the above-described method, capable of bindingto the cell surface of the prostate cancer cell and labelled with animaging agent under conditions permitting formation of a complex betweenthe ligand and the cell surface PSM′ antigen. This invention furtherprovides a composition comprising an effective imaging agent of the PSM′antigen ligand and a pharmaceutically acceptable carrier.Pharmaceutically acceptable carriers are well known to one of ordinaryskill in the art. For an example, such a pharmaceutically acceptablecarrier can be physiological saline.

Also provided by this invention is a purified mammalian PSM′ antigen. Asused herein, the term “purified alternatively spliced prostate-specificmembrane antigen” shall mean isolated naturally-occurringprostate-specific membrane antigen or protein (purified from nature ormanufactured such that the primary, secondary and tertiary conformation,and posttranslational modifications are identical to naturally-occurringmaterial) as well as non-naturally occurring polypeptides having aprimary structural conformation (i.e. continuous sequence of amino acidresidues). Such polypeptides include derivatives and analogs.

This invention provides an isolated nucleic acid comprising a promotersequence normally associated with the transcription of a gene encoding ahuman prostate-specific membrane antigen. In one embodiment regulatoryelements are set forth in FIG. 15. In another embodiment the promoter isbetween nucleotide −1 to −641 of FIG. 15A.

This invention provides a method to identify such natural ligand orother ligand which can bind to the PSM′ antigen. A method to identifythe ligand comprises a) coupling the purified mammalian PSM′ antigen toa solid matrix, b) incubating the coupled purified mammalian PSM′protein with the potential ligands under the conditions permittingbinding of ligands and the purified PSM′ antigen; c) washing the ligandand coupled purified mammalian PSM′ antigen complex formed in b) toeliminate the nonspecific binding and impurities and finally d) elutingthe ligand from the bound purified mammalian PSM′ antigen. Thetechniques of coupling proteins to a solid matrix are well known in theart. Potential ligands may either be deduced from the structure ofmammalian PSM′ by other empirical experiments known by ordinary skilledpractitioners. The conditions for binding may also easily be determinedand protocols for carrying such experimentation are known to thoseskilled in the art. The ligand-PSM′ antigen complex will be washed.Finally, the bound ligand is eluted and characterized. Standard ligandscharacterization techniques are well known in the art.

The above method may also be used to purify ligands from any biologicalsource. For purification of natural ligands in the cell, cell lysates,serum or other biological samples will be used to incubate with themammalian PSM′ antigen bound on a matrix. Specific natural ligand willthen be identified and purified as above described.

With the protein sequence information, antigenic areas may be identifiedand antibodies directed against these areas may be generated andtargeted to the prostate cancer for imaging the cancer or therapies.

This invention provides an antibody directed against the amino acidsequence of a mammalian PSM′ antigen.

This invention provides a method to select specific regions on the PSM′antigen to generate antibodies. The protein sequence may be determinedfrom the PSM′ DNA sequence. Amino acid sequences may be analyzed bymethods well known to those skilled in the art to determine whether theyproduce hydrophobic or hydrophilic regions in the proteins which theybuild. In the case of cell membrane proteins, hydrophobic regions arewell known to form the part of the protein that is inserted into thelipid bilayer of the cell membrane, while hydrophilic regions arelocated on the cell surface, in an aqueous environment. Usually, thehydrophilic regions will be more immunogenic than the hydrophobicregions. Therefore the hydrophilic amino acid sequences may be selectedand used to generate antibodies specific to mammalian PSM antigen. Foran example, hydrophilic sequences of the human PSM antigen shown inhydrophilicity plot may be easily selected. The selected peptides may beprepared using commercially available machines. As an alternative, DNA,such as a cDNA or a fragment thereof, may be cloned and expressed andthe resulting polypeptide recovered and used as an immunogen.

Polyclonal antibodies against these peptides may be produced byimmunizing animals using the selected peptides. Monoclonal antibodiesare prepared using hybridoma technology by fusing antibody producing Bcells from immunized animals with myeloma cells and selecting theresulting hybridoma cell line producing the desired antibody.Alternatively, monoclonal antibodies may be produced by in vitrotechniques known to a person of ordinary skill in the art. Theseantibodies are useful to detect the expression of mammalian PSM antigenin living animals, in humans, or in biological tissues or fluidsisolated from animals or humans.

In one embodiment, peptides Asp-Glu-Leu-Lys-Ala-Glu (SEQ ID No.),Asn-Glu-Asp-Gly-Asn-Glu (SEQ ID No.) and Lys-Ser-Pro-Asp-Glu-Gly (SEQ IDNo.) of human PSM antigen are selected.

This invention further provides polyclonal and monoclonal antibody(ies)against peptides Asp-Glu-Leu-Lys-Ala-Glu (SEQ ID No.),Asn-Glu-Asp-Gly-Asn-Glu (SEQ ID No.) and Lys-Ser-Pro-Asp-Glu-Gly (SEQ IDNo.).

This invention provides a method of imaging prostate cancer in humanpatients which comprises administering to the patient the monoclonalantibody directed against the peptide of the mammalian PSM′ antigencapable of binding to the cell surface of the prostate cancer cell andlabeled with an imaging agent under conditions permitting formation of acomplex between the monoclonal antibody and the cell surfaceprostate-specific membrane antigen. The imaging agent is a radioisotopesuch as Indium¹¹¹.

This invention further provides a prostate cancer specific imaging agentcomprising the antibody directed against PSM′ antigen and a radioisotopeconjugated thereto.

This invention also provides a composition comprising an effectiveimaging amount of the antibody directed against the PSM′ antigen and apharmaceutically acceptable carrier. The methods to determine effectiveimaging amounts are well known to a skilled practitioner. One method isby titration using different amounts of the antibody.

In addition to the standard pharmacophores that can be added to knowstructures, with the PSM transfectants one can identify potentialligands from combinatorial libraries that might not have been otherwisepredicted such combinatorial libraries can be synthetic, peptide, or RNAbased.

This invention further provides an immunoassay for measuring the amountof the prostate-specific membrane antigen in a biological samplecomprising steps of a) contacting the biological sample with at leastone antibody directed against the PSM′ antigen to form a complex withsaid antibody and the prostate-specific membrane antigen, and b)measuring the amount of the prostate-specific membrane antigen in saidbiological sample by measuring the amount of said complex. One exampleof the biological sample is a serum sample.

This invention provides a method to purify mammalian prostate-specificmembrane antigen comprising steps of a) coupling the antibody directedagainst the PSM′ antigen to a solid matrix; b) incubating the coupledantibody of a) with lysate containing prostate-specific membrane antigenunder the condition which the antibody and prostate membrane specificcan bind; c) washing the solid matrix to eliminate impurities and d)eluting the prostate-specific membrane antigen from the coupledantibody.

This invention also provides a transgenic nonhuman mammal whichcomprises the isolated nucleic acid molecule encoding a mammalian PSM′antigen. This invention further provides a transgenic nonhuman mammalwhose genome comprises antisense DNA complementary to DNA encoding amammalian prostate-specific membrane antigen so placed as to betranscribed into antisense mRNA complementary to mRNA encoding theprostate-specific membrane antigen and which hybridizes to mRNA encodingthe prostate specific antigen thereby reducing its translation.

Animal model systems which elucidate the physiological and behavioralroles of mammalian PSM′ antigen are produced by creating transgenicanimals in which the expression of the PSM′ antigen is either increasedor decreased, or the amino acid sequence of the expressed PSM antigen isaltered, by a variety of techniques. Examples of these techniquesinclude, but are not limited to: 1) Insertion of normal or mutantversions of DNA encoding a mammalian PSM′ antigen, by microinjection,electroporation, retroviral transfection or other means well known tothose skilled in the art, into appropriate fertilized embryos in orderto produce a transgenic animal or 2) Homologous recombination of mutantor normal, human or animal versions of these genes with the native genelocus in transgenic animals to alter the regulation of expression or thestructure of these PSM′ antigen sequences. The technique of homologousrecombination is well known in the art. It replaces the native gene withthe inserted gene and so is useful for producing an animal that cannotexpress native PSM antigen but does express, for example, an insertedmutant PSM antigen, which has replaced the native PSM antigen in theanimal's genome by recombination, resulting in under expression of thetransporter. Microinjection adds genes to the genome, but does notremove them, and so is useful for producing an animal which expressesits own and added PSM antigens, resulting in over expression of the PSMantigens.

One means available for producing a transgenic animal, with a mouse asan example, is as follows: Female mice are mated, and the resultingfertilized eggs are dissected out of their oviducts. The eggs are storedin an appropriate medium such as Me medium (16). DNA or cDNA encoding amammalian PSM antigen is purified from a vector by methods well known inthe art. Inducible promoters may be fused with the coding region of theDNA to provide an experimental means to regulate expression of thetrans-gene. Alternatively or in addition, tissue specific regulatoryelements may be fused with the coding region to permit tissue-specificexpression of the trans-gene. The DNA, in an appropriately bufferedsolution, is put into a microinjection needle (which may be made fromcapillary tubing using a pipet puller) and the egg to be injected is putin a depression slide. The needle is inserted into the pronucleus of theegg, and the DNA solution is injected. The injected egg is thentransferred into the oviduct of a pseudopregnant mouse (a mousestimulated by the appropriate hormones to maintain pregnancy but whichis not actually pregnant), where it proceeds to the uterus, implants,and develops to term. As noted above, microinjection is not the onlymethod for inserting DNA into the egg cell, and is used here only forexemplary purposes.

Another use of the PSM antigen sequence is to isolate homologous gene orgenes in different mammals. The gene or genes can be isolated by lowstringency screening of either cDNA or genomic libraries of differentmammals using probes from PSM sequence. The positive clones identifiedwill be further analyzed by DNA sequencing techniques which are wellknown to an ordinary person skilled in the art. For example, thedetection of members of the protein serine kinase family by homologyprobing.

This invention provides a method of suppressing or modulating metastaticability of prostate tumor cells, prostate tumor growth or elimination ofprostate tumor cells comprising introducing a DNA molecule encoding analternatively spliced prostate specific membrane antigen operativelylinked to a 5′ regulatory element into a tumor cell of a subject, in away that expression of the alternatively spliced prostate specificmembrane antigen is under the control of the regulatory element, therebysuppressing or modulating metastatic ability of prostate tumor cells,prostate tumor growth or elimination of prostate tumor cells. Thesubject may be a mammal or more specifically a human.

In one embodiment, the DNA molecule is operatively linked to a 5′regulatory element forms part of a transfer vector which is insertedinto a cell or organism. In addition the vector is capable orreplication and expression of the alternatively spliced prostatespecific membrane antigen. The DNA molecule can be integrated into agenome of a eukaryotic or prokaryotic cell or in a host cell containingand/or expressing an alternatively spliced prostate specific membraneantigen.

Further, the DNA molecule encoding alternatively spliced prostatespecific membrane antigen may be introduced by a bacterial, viral,fungal, animal, or liposomal delivery vehicle. Other means are alsoavailable and known to an ordinary skilled practitioner.

Further, the DNA molecule encoding an alternatively spliced prostatespecific membrane antigen operatively linked to a promoter or enhancer.A number of viral vectors have been described including those made fromvarious promoters and other regulatory elements derived from virussources. Promoters consist of short arrays of nucleic acid sequencesthat interact specifically with cellular proteins involved intranscription. The combination of different recognition sequences andthe cellular concentration of the cognate transcription factorsdetermines the efficiency with which a gene is transcribed in aparticular cell type.

Examples of suitable promoters include a viral promoter. Viral promotersinclude: adenovirus promoter, an simian virus 40 (SV40) promoter, acytomegalovirus (CMV) promoter, a mouse mammary tumor virus (MMTV)promoter, a Malony murine leukemia virus promoter, a murine sarcomavirus promoter, and a Rous sarcoma virus promoter.

Further, another suitable promoter is a heat shock promoter.Additionally, a suitable promoter is a bacteriophage promoter. Examplesof suitable bacteriophage promoters include but not limited to, a T7promoter, a T3 promoter, an SP6 promoter, a lambda promoter, abaculovirus promoter.

Also suitable as a promoter is an animal cell promoter such as aninterferon promoter, a metallothionein promoter, an immunoglobulinpromoter. A fungal promoter is also a suitable promoter. Examples offungal promoters include but are not limited to, an ADC1 promoter, anARG promoter, an ADH promoter, a CYC1 promoter, a CUP promoter, an ENO1promoter, a GAL promoter, a PHO promoter, a PGK promoter, a GAPDHpromoter, a mating type factor promoter. Further, plant cell promotersand insect cell promoters are also suitable for the methods describedherein.

This invention provides a method of suppressing or modulating metastaticability of prostate tumor cells, prostate tumor growth or elimination ofprostate tumor cells, comprising introducing a DNA molecule encoding analternatively spliced prostate specific membrane antigen operativelylinked to a 5′ regulatory element coupled with a therapeutic DNA into atumor cell of a subject, thereby suppressing or modulating metastaticability of prostate tumor cells, prostate tumor growth or elimination ofprostate tumor cells. The subject may be a mammal or more specifically ahuman.

Further, the therapeutic DNA which is coupled to the DNA moleculeencoding a prostate specific membrane antigen operatively linked to a 5′regulatory element into a tumor cell may code for a cytokine, viralantigen, or a pro-drug activating enzyme. Other means are also availableand known to an ordinary skilled practitioner.

In addition, this invention provides a prostate tumor cell, comprising aDNA molecule isolated from mammalian nucleic acid encoding analternatively spliced mammalian prostate-specific membrane antigen underthe control a 5′ regulatory element.

As used herein, DNA molecules include complementary DNA (cDNA),synthetic DNA, and genomic DNA.

This invention provides a therapeutic vaccine for preventing humanprostate tumor growth or stimulation of prostate tumor cells in asubject, comprising administering an effective amount to the prostatecell, and a pharmaceutical acceptable carrier, thereby preventing thetumor growth or stimulation of tumor cells in the subject. Other meansare also available and known to an ordinary skilled practitioner.

This invention provides a method of detecting hematogenous micrometastictumor cells of a subject, comprising (A) performing nested polymerasechain reaction (PCR) on blood, bone marrow or lymph node samples of thesubject using the prostate specific membrane antigen primers oralternatively spliced prostate specific antigen primers, and (B)verifying micrometastases by DNA sequencing and Southern analysis,thereby detecting hematogenous micrometastic tumor cells of the subject.The subject may be a mammal or more specifically a human.

The micrometastatic tumor cell may be a prostatic cancer and the DNAprimers may be derived from prostate specific antigen. Further, thesubject may be administered with simultaneously an effective amount ofhormones, so as to increase expression of prostate specific membraneantigen. Further, growth factors or cytokine may be administered inseparately or in conjunction with hormones. Cytokines include, but arenot limited to: transforming growth factor beta, epidermal growth factor(EGF) family, fibroblast growth factors, hepatocyte growth factor,insulin-like growth factors, B-nerve growth factor, platelet-derivedgrowth factor, vascular endothelial growth factor, interleukin 1, IL-1receptor antagonist, interleukin 2, interleukin 3, interleukin 4,interleukin 5, interleukin 6, IL-6 soluble receptor, interleukin 7,interleukin 8, interleukin 9, interleukin 10, interleukin 11,interleukin 12, interleukin 13, angiogenin, chemokines, colonystimulating factors, granulocyte-macrophage colony stimulating factors,erythropoietin, interferon, interferon gamma, leukemia inhibitoryfactor, oncostatin M, pleiotrophin, secretory leukocyte proteaseinhibitor, stem cell factor, tumor necrosis factors, adhesion molecule,and soluble tumor necrosis factor (TNF) receptors.

This invention provides a method of abrogating the mitogenic responsedue to transferrin, comprising introducing a DNA molecule encodingprostate specific membrane antigen operatively linked to a 5′ regulatoryelement into a tumor cell, the expression of which gene is directlyassociated with a defined pathological effect within a multicellularorganism, thereby abrogating mitogen response due to transferrin. Thetumor cell may be a prostate cell.

This invention provides a method of determining prostate cancerprogression in a subject which comprises: a) obtaining a suitableprostate tissue sample; b) extracting RNA from the prostate tissuesample; c) performing a RNAse protection assay on the RNA therebyforming a duplex RNA-RNA hybrid; d) detecting PSM and PSM′ amounts inthe tissue sample; e) calculating a PSM/PSM′ tumor index, therebydetermining prostate cancer progression in the subject. In-situhyribridization may be performed in conjunction with the above detectionmethod.

This invention provides a method of detecting prostate cancer in asubject which comprises: (a) obtaining from a subject a prostate tissuesample; (b) treating the tissue sample so as to separately recovernucleic acid molecules present in the prostate tissue sample; (c)contacting the resulting nucleic acid molecules with multiple pairs ofsingle-stranded labeled oligonucleotide primers, each such pair beingcapable of specifically hybridizing to the tissue sample, underhybridizing conditions; (d) amplifying any nucleic acid molecules towhich a pair of primers hybridizes so as to obtain a double-strandedamplification product; (e) treating any such double-strandedamplification product so as to obtain single-stranded nucleic acidmolecules therefrom; (f) contacting any resulting single-strandednucleic acid molecules with multiple single-stranded labeledoligonucleotide probes, each such probe containing the same label andbeing capable of specifically hybridizing with such tissue sample, underhybridizing conditions; (g) contacting any resulting hybrids with anantibody to which a marker is attached and which is capable ofspecifically forming a complex with the labeled-probe, when the probe ispresent in such a complex, under complexing conditions; and (h)detecting the presence of any resulting complexes, the presence thereofbeing indicative of prostate cancer in a subject.

This invention provides a method of enhancing antibody based targetingof PSM′ in prostate tissue for diagnosis or therapy of prostate cancercomprising administering to a patient b-FGF in sufficient amount tocause upregulation of PSM′ expression.

This invention provides a method of enhancing antibody based targetingof PSM′ in prostate tissue for diagnosis or therapy of prostate cancercomprising administering to a patient TGF in sufficient amount to causeupregulation of PSM expression or PSM′.

This invention provides a method of enhancing antibody based targetingof PSM′ in prostate tissue for diagnosis or therapy of prostate cancercomprising administering to a patient EGF in sufficient amount to causeupregulation of PSM′ expression.

This invention provides a method of detecting in a sample the presenceof a nucleic acid encoding an alternatively spliced humanprostate-specific membrane antigen which comprises: a) obtaining asuitable sample; b) extracting RNA from the sample; c) contacting theRNA with reverse transcriptase under suitable conditions to obtain acDNA; d) contacting the cDNA under hybridizing conditions with twooligonucleotide primers, i) the first primer being capable ofspecifically hybridizing to a sequence within a DNA sequence encodingprostate specific membrane antigen located immediately 3′ of nucleotide114 of such DNA sequence, with the proviso that the 3′ end of the primerdoes not hybridize to any sequence located 5′ of nucleotide 114, and ii)the second primer being capable of specifically hybridizing to asequence within a DNA sequence encoding prostate specific membraneantigen located immediately 5′ of nucleotide 381 of such DNA sequence,with the proviso that the 5′ end of the primer does not hybridize to anysequence located 3′ of nucleotide 381; d) amplifying any cDNA to whichthe primers hybridize to so as to obtain amplification product; e)determining the size of the amplification product; f) comparing the sizeof the amplification product to the size of the amplification productknown to be obtained using the same primers with a non alternativelyspliced human prostate specific membrane antigen, wherein a smalleramplification product is indicative of the presence of the alternativelyspliced prostate specific membrane antigen, so as to thereby detect thepresence of the alternatively spliced human prostate-specific membraneantigen in the sample.

In one embodiment the suitable sample may be any bodily tissue of fluidwhich includes but is not limited to: blood, bone marrow, and lymphnodes.

In one embodiment the primers are at least 14-25 nucleotides in length.In another embodiment the primers are at least 15 nucleotide in length.In another embodiment the primers are 15 nucleotides in length. Inanother embodiment multiple primers are used. Construction of primerswhich hybridize and hybridizing conditions are known to those skilled inthe art. For example, based on FIG. 18 one skilled in the art mayconstruct primers which hybridize to the prostate specific membraneantigen before nucleotide 114 and after nucleotide 381.

Further, a method of determining the amount of the amplification productor products (i.e. 2 or more bands) as well as the ratio of each productis known to those skilled in the art. For example, the amount ofprostate specific membrane antigen or alternatively spliced prostatespecific membrane antigen may be determined by density, bindingradiolabled probes, autoradiography, UV spectrography,spectrophotometer, optical scan, and phospho-imaging.

This invention provides a method of detecting a prostate tumor cell in asubject which comprises: which comprises: a) obtaining a suitablesample; b) extracting RNA from the sample; c) contacting the RNA withreverse transcriptase under suitable conditions to obtain a cDNA; d)contacting the cDNA under hybridizing conditions with twooligonucleotide primers, i) the first primer being capable ofspecifically hybridizing to a sequence within a DNA sequence encodingprostate specific membrane antigen located immediately 3′ of nucleotide114 of such DNA sequence, with the proviso that the 3′ end of the primerdoes not hybridize to any sequence located 5′ of nucleotide 114, and ii)the second primer being capable of specifically hybridizing to asequence within a DNA sequence encoding prostate specific membraneantigen located immediately 5′ of nucleotide 381 of such DNA sequence,with the proviso that the 5′ end of the primer does not hybridize to anysequence located 3′ of nucleotide 381; d) amplifying any cDNA to whichthe primers hybridize to so as to obtain amplification product; e)determining the amount of the amplification product; f) comparing theamount of the amplification product to the amount of the amplificationproduct known to be obtained using the same primers with a nonalternatively spliced human prostate specific membrane antigen, whereina greater amount of the prostate specific membrane antigen is indicativeof a prostate tumor cell in the subject, so as to thereby detectprostate tumor cell in the subject.

In PCR techniques, oligonucleotide primers complementary to the two 3′borders of the DNA of the prostate specific membrane (PSM) antigen to beamplified are synthesized. The polymerase chain reaction is then carriedout using the two primers. See PCR Protocols: A Guide to Methods andApplications. Hybridization of PSM antigen DNA to the above nucleic acidprobes can be performed by a Southern blot under stringent hybridizationconditions as described herein.

Oligonucleotides for use as probes or PCR primers are chemicallysynthesized according to the solid phase phosphoramidite triester methodfirst described by Beaucage and Carruthers using an automatedsynthesizer, as described in Needham-VanDevanter. Purification ofoligonucleotides is by either native acrylamide gel electrophoresis orby anion-exchange HPLC as described in Pearson, J. D. and Regnier, F. E.The sequence of the synthetic oligonucleotide can be verified using thechemical degradation method of Maxam, A. M. and Gilbert, W.

Accepted means for conducting hybridization assays are known and generaloverviews of the technology can be had from a review of: Nucleic AcidHybridization: A Practical Approach; Hybridization of Nucleic AcidsImmobilized on Solid Supports; Analytical Biochemistry and Innis et al.,PCR Protocols.

If PCR is used in conjunction with nucleic acid hybridization, primersare designed to target a specific portion of the nucleic acid of DNA ofthe PSM antigen. From the information provided herein, those of skill inthe art will be able to select appropriate specific primers.

It will be apparent to those of ordinary skill in the art that aconvenient method for determining whether a probe is specific for PSMantigen or PSM′ antigen utilizes a Southern blot (or Dot blot). Briefly,to identify a target specific probe DNA is isolated from the PSM or PSM′antigen. Test DNA is transferred to a solid (e.g., charged nylon)matrix. The probes are labelled following conventional methods.Following denaturation and/or prehybridization steps known in the art,the probe is hybridized to the immobilized DNAs under stringentconditions. Stringent hybridization conditions will depend on the probeused and can be estimated from the calculated T_(m) (meltingtemperature) of the hybridized probe (see, e.g., Sambrook for adescription of calculation of the T_(m)). For radioactively-labeled DNAor RNA probes an example of stringent hybridization conditions ishybridization in a solution containing denatured probe and 5×SSC at 65°C. for 8-24 hours followed by washes in 0.1×SSC, 0.1% SDS (sodiumdodecyl sulfate) at 50-65° C. In general, the temperature and saltconcentration are chosen so that the post hybridization wash occurs at atemperature that is about 5° C. below the T_(M) of the hybrid. Thus fora particular salt concentration the temperature may be selected that is5° C. below the T_(M) or conversely, for a particular temperature, thesalt concentration is chosen to provide a T_(M) for the hybrid that is5° C. warmer than the wash temperature. Following stringenthybridization and washing, a probe that hybridizes to the PSM antigen orPSM′ antigen as evidenced by the presence of a signal associated withthe appropriate target and the absence of a signal from the non-targetnucleic acids, is identified as specific. It is further appreciated thatin determining probe specificity and in utilizing the method of thisinvention a certain amount of background signal is typical and caneasily be distinguished by one of skill from a specific signal. Two foldsignal over background is acceptable.

This invention provides a therapeutic agent comprising antibodies orligand(s) directed against PSM′ antigen and a cytotoxic agent conjugatedthereto or antibodies linked enzymes which activate prodrug to kill thetumor. The cytotoxic agent may either be a radioisotope or toxin.

This invention provides a compound comprising a conjugate of a cytotoxicagent and one or more amino acid residues, wherein each amino acidresidue is glutamate or aspartate. In one embodiment the amino acidresidues alternate.

Examples of cytotoxic chemotherapeutic agents or antineolastic agents)include, but are not limited to the following: Antimetaboloites:Denopterin, Edatrexate, Piritrexim, Pteropterin, Tomudex, Tremetrexate,Cladribine, Fludarabine, 6-Mercaptopurine, Thiamiprine, Thioguanine,Ancitabine, Azacitidine, 6-Azauridine, Carmofur, Cytarabine,Doxifluride, Emitefur, Enocitabine, Floxuridine, Fluoroucit,Gemcitabine, and Tegafur.

Alkaloids: Docetaxel, Etoposide, Irinotecan, Paclitaxel, Teniposide,Topotecan, VinblastinE, Vincristine, and Vindesine.

Alkylating agents: Alkyl Sulfonates: Busulfan, Improsulfan, Piposulfan,Aziridines, Benzodepa, Carboquone, Meuredepa, Uredepa, Ethylenimines andMethylmelamines, Altretamine, Triethylenemelamine,Triethylenophosphoramide, Triethylenethiophosphoramide, Chlorambucil,Chlornaphazine, Cyclophosphamide, Estramustine, Ifosfamide,Mechlorethamine, Mechlorethamine Oxide Hydrochloride, Melphalan,Novembiechin, Perfosfamide, Phenesterine, Prednimustine, Trofosfamide,Uracil Mustard, Carmustine, Chlorozotocin, Fotemustine, Lomustine,Nimustine, Ranimustine, Dacarbazine, Mannomustine, Mitbronitol,Mitolactol, Pipobroman, Temozolomide, Antibiotics and Analogs:Aclacinomycins, Actinomycin, Anthramycin, Azaserine, Bleomycins,Cactinomycin, Carubicin, Carzinophilin, Chromomycins, Dactinomycin,Caunorubicin, 6-Diazo-5-oxo-L-norleucine, Doxorubicin, Epirubicin,Idarubicin, Menogaril, Mitomycins, Mycophenolic Acid, Nogalamycin,Olivomycins, Peplomycin, Pirarubicin, Plicamycin, Porfiromycin,Puromycin, Streptonigrin, Streptozocin, Tubercidin, Zinostatin,Zorubicin, and L-Asparaginase.

Immunodulators: Interferon, Interferon-B, Interferon-Y, Interleukin-2,Lentinan, Propagermanium, PSK, Roquinimex, Sizofran, and Ubenimex.Platinum complexes: Carboplatin, Cisplatin, Miboplatin, and Oxaliplatin.

Others: Aceglatone, Amsacrine, Bisantrene, Defoosfamide, Demecolcine,Diaziqone, Eflornithine, Eliptinium Acetate, Etoglucid, Fenertinide,Gallium Nitrate, Hydroxyurea, Lonidamine, Miltefosine, Mitoguazone,Mitoxantrone, Mopidamol, Nitracirine, Pentostatin, Phenamet,Podophyllinic Acid 2-Ethyl-hydrazide, Procarbazine, Razoxane,Sobuzoxane, Spirogermanium, Tenuazonic Acid, Triaziquone, Urethan,Calusterone, Dromostanolone, Epitiostanol, Mepitiostane, Testolactone,Amiglutehimide, Mitotane, Trilostane, Droloxifene, Tamoxifen,Toremifene, Aminoglutethimide, Anastrozole, Fadrozole, Formestane,Letrozole, Fosfestrol, Hexestrol, Polyestradiol Phosphate, Buserlin,Goserlin, Leuprolide, Triptorelin, Chlormadinone Acetate,Medroxyprogesterone, Megerstrol Acetate, Melengestrol, Porfimer Sodium,Americium, Chromic Phosphate, Radioactive Cobalt, I-Ehtiodized Oil,Gold, Radioactive, Colloidal, Iobenguane: Radium, Radon, Sodium Iodide,Sodium Phosphate, Radioactive, Batimastat, Folinic Acid, Amifostine,Etanidazole, Etamidozole, and Mesna.

This invention provides a compound, wherein the compound has thestructure:

-   -   wherein n is an integer from 1-10 inclusive.

In one embodiment glutamate may be in L or D to form either4-amino-N¹⁰-methyl pteroyl-L-glutamate or 4-amino-N¹⁰-methylpteroyl-D-glutamate. In another embodiment aspartate may substitute theglutamate to form 4-amino-N¹⁰-methyl pteroyl-L-aspartate. In anotherembodiment aspartate may substitute the glutamate to form4-amino-N¹⁰-methyl pteroyl-D-aspartate. In another embodiment the4′-amino-N¹⁰-methyl pteroyl may have alternating glutamate or aspartatmoieties. The glutamate or aspartate are bound to the methotrexate atthe alpha carbon position of methotrexate.

This invention provides a compound, wherein the compound has thestructure:

-   -   wherein n is an integer from 1-10 inclusive.

In one embodiment glutamate may be in the L or D to form eitherN-phosphonoacetyl-L-aspartyl (PALA)-glutamate orN-phsophonoacetyl-D-aspartyl-glutamate. In another embodiment aspartatemay substitute the glutamate to formN-phsophonoacetyl-L-aspartyl-aspartate. In another embodiment the4-amino-N¹⁰-methyl pteroyl may have alternating glutamate or aspartatemoieties.

This invention provides a compound, wherein the compound has thestructure:

-   -   wherein n is an integer from 1-10 inclusive.

In one embodiment glutamate may be in the L or D to form either4-amino-10-ethyl-10-deazapteroyl (EDAM)-L-glutamate or4-amino-10-ethyl-10-deazapteroyl-D-glutamate. In another embodimentaspartate may substitute the glutamate to form4-amino-10-ethyl-10-deazapteroyl-L-aspartate. In another embodiment the4-amino-10-ethyl-10-deazapteroyl may have alternating glutamate oraspartat moieties.

This invention provides a pharmaceutical composition comprising any ofthe above compounds in a therapeutically effective amount and apharmaceutically acceptable carrier.

This invention provides a method of making prostate cells susceptible toa cytotoxic agent, which comprises contacting the prostate cells withany of the above compounds in an amount effective to render the prostatecells susceptible to the cytotoxic chemotherapeutic agent.

This invention provides a pharmaceutical composition comprising aneffective amount the alternatively spliced. PSM′ and a carrier ordiluent. Further, this invention provides a method for administering toa subject, preferably a human, the pharmaceutical composition. Further,this invention provides a composition comprising an amount of thealternatively spliced PSM′ and a carrier or diluent. Specifically, thisinvention may be used as a food additive.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. Precise amountsof active ingredient required to be administered depend on the judgmentof the practitioner and are peculiar to each subject.

In one embodiment the therapeutic effective amount is 100-10,000 mg/m²IV with rescue. In another embodiment the therapeutic effective amountis 300-1000 mg/m² IV or continuous infusion. In another embodiment thetherapeutic effective amount is 100 mg/m² IV continuous infusion. Inanother embodiment the therapeutic effective amount is 40-75 mg/m²rapidly. In another embodiment the therapeutic effective amount is 30mg/m² for 3 days by continuous IV.

Suitable regimes for initial administration and booster shots are alsovariable, but are typified by an initial administration followed byrepeated doses at one or more hour intervals by a subsequent injectionor other administration.

As used herein administration means a method of administering to asubject. Such methods are well known to those skilled in the art andinclude, but are not limited to, administration topically, parenterally,orally, intravenously, intramuscularly, subcutaneously or by aerosol.Administration of PSM may be effected continuously or intermittently.

The pharmaceutical formulations or compositions of this invention may bein the dosage form of solid, semi-solid, or liquid such as, e.g.,suspensions, aerosols or the like. Preferably the compositions areadministered in unit dosage forms suitable for single administration ofprecise dosage amounts. The compositions may also include, depending onthe formulation desired, pharmaceutically-acceptable, non-toxic carriersor diluents, which are defined as vehicles commonly used to formulatepharmaceutical compositions for animal or human administration. Thediluent is selected so as not to affect the biological activity of thecombination. Examples of such diluents are distilled water,physiological saline, Ringer's solution, dextrose solution, and Hank'ssolution. In addition, the pharmaceutical composition or formulation mayalso include other carriers, adjuvants; or nontoxic, nontherapeutic,nonimmunogenic stabilizers and the like. Effective amounts of suchdiluent or carrier are those amounts which are effective to obtain apharmaceutically acceptable formulation in terms of solubility ofcomponents, or biological activity, etc

This invention also provides a method of detecting a subject with cancercomprising a) contacting a cell of the neo-vasculature of a subject witha ligand which binds to the extraccelular domain of the PSM antigenunder conditions permitting formation of a complex; and b) detecting thecomplex with a labelled imaging agent, thereby detecting a subject withcancer.

In one embodiment the cancer is, but is not limited to: kidney, colon,or bladder. In one embodiment the ligand is CYT-356. In anotherembodiment the ligand is any antibody, monoclonal or polyclonal whichbinds to the extracellular domain of PSM antigen. In one embodiment thecells of endothelial cells of the neo-vasculature of a subject withcancer.

This invention will be better understood from the Experimental Detailswhich follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims which followthereafter.

EXPERIMENTAL DETAILS Example 1 Expression of the Prostate SpecificMembrane Antigen

A 2.65 kb complementary DNA encoding PSM was cloned. Immunohistochemicalanalysis of the LNCaP, DU-145, and PC-3 prostate cancer cell lines forPSM expression using the 7E11-C5.3 antibody reveals intense staining inthe LNCaP cells, with no detectable expression in both the DU-145 andPC-3 cells. Coupled in-vitro transcription/translation of the 2.65 kbfull-length PSM cDNA yields an 84 kDa protein corresponding to thepredicted polypeptide molecular weight of PSM. Post-translationalmodification of this protein with pancreatic canine microsomes yieldsthe expected 100 kDa PSM antigen. Following transfection of PC-3 cellswith the full-length PSM cDNA in a eukaryotic expression vectorapplicant's detect expression of the PSM glycoprotein by Westernanalysis using the 7E11-C5.3 monoclonal antibody. Ribonucleaseprotection analysis demonstrates that the expression of PSM mRNA isalmost entirely prostate-specific in human tissues. PSM expressionappears to be highest in hormone-deprived states and is hormonallymodulated by steroids, with DHT down regulating PSM expression in thehuman prostate cancer cell line LNCaP by 8-10 fold, testosterone downregulating PSM by 3-4 fold, and corticosteroids showing no significanteffect. Normal and malignant prostatic tissues consistently show highPSM expression, whereas heterogeneous, and at times absent, fromexpression of PSM in benign prostatic hyperplasia. LNCaP tumorsimplanted and grown both orthotopically and subcutaneously in nude mice,abundantly express PSM providing an excellent in-vivo model system tostudy the regulation and modulation of PSM expression.

Materials and Methods:

Cells and Reagents: The LNCaP, DU-145, and PC-3 cell lines were obtainedfrom the American Type Culture Collection. Details regarding theestablishment and characteristics of these cell lines have beenpreviously published. Unless specified otherwise, LNCaP cells were grownin RPMI 1640 media supplemented with L-glutamine, nonessential aminoacids, and 5% fetal calf serum (Gibco-BRL, Gaithersburg, Md.) in a CO₂incubator at 37 C. DU-145 and PC-3 cells were grown in minimal essentialmedium supplemented with 10% fetal calf serum. All cell media wereobtained from the MSKCC Media Preparation Facility. Restriction andmodifying enzymes were purchased from Gibco-BRL unless otherwisespecified.

Immunohistochemical Detection of PSM: Avidin-biotin method of detectionwas employed to analyze prostate cancer cell lines for PSM antigenexpression. Cell cytospins were made on glass slides using 5×10⁴cells/100 ul per slide. Slides were washed twice with PBS and thenincubated with the appropriate suppressor serum for 20 minutes. Thesuppressor serum was drained off and the cells were incubated withdiluted 7E11-C5.3 (5 g/ml) monoclonal antibody for 1 hour. Samples werethen washed with PBS and sequentially incubated with secondaryantibodies for 30 minutes and with avidin-biotin complexes for 30minutes. Diaminobenzidine served as the chromogen and color developmentfollowed by hematoxylin counterstaining and mounting. Duplicate cellcytospins were used as controls for each experiment. As a positivecontrol, the anti-cytokeratin monoclonal antibody CAM 5.2 was usedfollowing the same procedure described above. Human EJ bladder carcinomacells served as a negative control.

In-Vitro Transcription/Translation of PSM Antigen: Plasmid 55Acontaining the full length 2.65 kb PSM cDNA in the plasmid pSPORT 1(Gibco-BRL) was transcribed in-vitro using the Promega TNT system(Promega Corp. Madison, Wis.). T7 RNA polymerase was added to the cDNAin a reaction mixture containing rabbit reticulocyte lysate, an aminoacid mixture lacking methionine, buffer, and ³⁵S-Methionine (Amersham)and incubated at 30 C for 90 minutes. Post-translational modification ofthe resulting protein was accomplished by the addition of pancreaticcanine microsomes into the reaction mixture (Promega Corp. Madison,Wis.). Protein products were analyzed by electrophoresis on 10% SDS-PAGEgels which were subsequently treated with Amplify autoradiographyenhancer (Amersham, Arlington Heights, Ill.) according to themanufacturers instructions and dried at 80 C in a vacuum dryer. Gelswere autoradiographed overnight at −70 C using Hyperfilm MP (Amersham).

Transfection of PSM into PC-3 Cells: The full length PSM cDNA wassubcloned into the pREP7 eukaryotic expression vector (Invitrogen, SanDiego, Calif.). Plasmid DNA was purified from transformed DH5-alphabacteria (Gibco-BRL) using Qiagen maxi-prep plasmid isolation columns(Qiagen Inc., Chatsworth, Calif.). Purified plasmid DNA (6-10 g) wasdiluted with 900 ul of Optimem media (Gibco-BRL) and mixed with 30 ul ofLipofectin reagent (Gibco-BRL) which had been previously diluted with9001 of Optimem media. This mixture was added to T-75 flasks of 40-50%confluent PC-3 cells in Optimem media. After 24-36 hours, cells weretrypsinized and split into 100 mm dishes containing RPMI 1640 mediasupplemented with 10% fetal calf serum and 1 mg/ml of Hygromycin B(Calbiochem, La. Jolla, Calif.). The dose of Hygromycin B used waspreviously determined by a time course/dose response cytotoxicity assay.Cells were maintained in this media for 2-3 weeks with changes of mediaand Hygromycin B every 4-5 days until discrete colonies appeared.Colonies were isolated using 6 mm cloning cylinders and expanded in thesame media. As a control, PC-3 cells were also transfected with thepREP7 plasmid alone. RNA was isolated from the transfected cells and PSMmRNA expression was detected by both RNase Protection analysis(described later) and by Northern analysis.

Western Blot Detection of PSM Expression: Crude protein lysates wereisolated from LNCaP, PC-3, and PSM-transfected PC-3 cells as previouslydescribed. LNCaP cell membranes were also isolated according topublished methods. Protein concentrations were quantitated by theBradford method using the BioRad protein reagent kit (BioRad, Richmond,Calif.). Following denaturation, 20 μg of protein was electrophoresed ona 10% SDS-PAGE gel at 25 mA for 4 hours. Gels were electroblotted ontoImmobilon P membranes (Millipore, Bedford, Mass.) overnight at 4 C.Membranes were blocked in 0.15M NaCl/0.01M Tris-HCl (TS) plus 5% BSAfollowed by a 1 hour incubation with 7E11-C5.3 monoclonal antibody (10μg/ml). Blots were washed 4 times with 0.15M NaCl/0.01M Tris-HCl/0.05%Triton-X 100 (TS-X) and incubated for 1 hour with rabbit anti-mouse IgG(Accurate Scientific, Westbury, N.Y.) at a concentration of 10 μg/ml.

Blots were then washed 4 times with TS-X and labeled with ¹²⁵I-Protein A(Amersham, Arlington Heights, Ill.) at a concentration of 1 millioncpm/ml. Blots were then washed 4 times with TS-X and dried on Whatman3MM paper, followed by overnight autoradiography at −70 C usingHyperfilm MP (Amersham).

Orthotopic and Subcutaneous LNCaP Tumor Growth in Nude Mice: LNCaP cellswere harvested from sub-confluent cultures by a one minute exposure to asolution of 0.25% trypsin and 0.02% EDTA. Cells were resuspended in RPMI1640 media with 5% fetal bovine serum, washed and diluted in eitherMatrigel (Collaborative Biomedical Products, Bedford, Mass.) or calciumand magnesium-free Hank's balanced salt solution (HESS). Only singlecell suspensions with greater than 90% viability by trypan blueexclusion were used for in vivo injection. Male athymic Swiss (nu/nu)nude mice 4-6 weeks of age were obtained from the MemorialSloan-Kettering Cancer Center Animal Facility. For subcutaneous tumorcell injection one million LNCaP cells resuspended in 0.2 mls. ofMatrigel were injected into the hindlimb of each mouse using adisposable syringe fitted with a 28 gauge needle. For orthotopicinjection, mice were first anesthetized with an intraperitonealinjection of Pentobarbital and placed in the supine position. Theabdomen was cleansed with Betadine and the prostate was exposed througha midline incision. 2.5 million LNCaP tumor cells in 0.1 ml. wereinjected directly into either posterior lobe using a 1 ml disposablesyringe and a 28 gauge needle. LNCaP cells with and without Matrigelwere injected. Abdominal closure was achieved in one layer usingAutoclip wound clips (Clay Adams, Parsippany, N.J.). Tumors wereharvested in 6-8 weeks, confirmed histologically by faculty of theMemorial Sloan-Kettering Cancer Center Pathology Department, and frozenin liquid nitrogen for subsequent RNA isolation.

RNA Isolation: Total cellular RNA was isolated from cells and tissues bystandard techniques (3 and 17) as well as by using RMAzol B(Cinna/Biotecx, Houston, Tex.). RNA concentrations and quality wereassessed by UV spectroscopy on a Beckman DU 640 spectrophotometer and bygel analysis. Human tissue total RNA samples were purchased fromClontech Laboratories, Inc., Palo Alto, Calif.

Ribonuclease Protection Assays: A portion of the PSM cDNA was subclonedinto the plasmid vector pSPORT 1 (Gibco-BRL) and the orientation of thecDNA insert relative to the flanking T7 and SP6 RNA polymerase promoterswas verified by restriction analysis. Linearization of this plasmidupstream of the PSM insert followed by transcription with SP6 RNApolymerase yields a 400 nucleotide antisense RNA probe, of which 350nucleotides should be protected from RNase digestion by PSM RNA. Thisprobe was used in FIG. 20. Plasmid IN-20, containing a 1 kb partial PSMcDNA in the plasmid pCR II (Invitrogen) was also used for riboprobesynthesis. IN-20 linearized with Xmn I (Gibco-BRL) yields a 298nucleotide anti-sense RNA probe when transcribed using SP6 RNApolymerase, of which 260 nucleotides should be protected from RNasedigestion by PSM mRNA. This probe was used in FIGS. 21 and 22. Probeswere synthesized using SP6 RNA polymerase (Gibco-BRL), rNTPs(Gibco-BRL), RNAsin (Promega), and ³²P-rCTP (NEN, Wilmington, Del.)according to published protocols (44). Probes were purified over NENSORB20 purification columns (NEN) and approximately 1 million cpm ofpurified, radiolabeled PSM probe was mixed with 10μ of each RNA andhybridized overnight at 45 C using buffers and reagents from the RPA IIkit (Ambion, Austin, Tex.). Samples were processed as per manufacturer'sinstructions and analyzed on 5% polyacrilamide/7M urea denaturing gelsusing Seq ACRYL reagents (ISS, Natick, Mass.). Gels were pre-heated to55 C and run for approximately 1-2 hours at 25 watts. Gels were thenfixed for 30 minutes in 10% methanol/10% acetic acid, dried onto Whatman3MM paper at 80 C in a BioRad vacuum dryer and autoradiographedovernight with Hyperfilm MP (Amersham). Quantitation of PSM expressionwas determined by using a scanning laser densitometer (LKB, Piscataway,N.J.).

Steroid Modulation Experiment: LNCaP cells (2 million) were plated ontoT-75 flasks in RPMI 1640 media supplemented with 5% fetal calf serum andgrown 24 hours until approximately 30-40% confluent. Flasks were thenwashed several times with phosphate-buffered saline and RPMI mediumsupplemented with 5% charcoal-extracted serum was added. Cells were thengrown for another 24 hours, at which time dihydrotesterone,testosterone, estradiol, progesterone, and dexamethasone (SteraloidsInc., Wilton, N.H.) were added at a final concentration of 2 nM. Cellswere grown for another 24 hours and RNA was then harvested as previouslydescribed and PSM expression analyzed by ribonuclease protectionanalysis.

Experimental Results

Immunohistochemical Detection of PSM: Using the 7E11-C5.3 anti-PSMmonoclonal antibody, PSM expression is clearly detectable in the LNCaPprostate cancer cell line, but not in the PC-3 and DU-145 cell lines(FIGS. 17A-17C): All normal and malignant prostatic tissues analyzedstained positively for PSM expression.

In-Vitro Transcription/Translation of PSM Antigen: As shown in FIG. 18,coupled in-vitro transcription/translation of the 2.65 kb full-lengthPSM cDNA yields an 84 kDa protein species in agreement with the expectedprotein product from the 750 amino acid PSM open reading frame.Following post-translational modification using pancreatic caninemicrosomes were obtained a 100 kDa glycosylated protein speciesconsistent with the mature, native PSM antigen.

Detection of PSM Antigen in LNCaP Cell Membranes and Transfected PC-3Cells: PC-3 cells transfected with the full length PSM cDNA in the pREP7expression vector were assayed for expression of SM mRNA by Northernanalysis. A clone with high PSM mRNA expression was selected for PSMantigen analysis by Western blotting using the 7E11-C5.3 antibody. InFIG. 19, the 100 kDa PSM antigen is well expressed in LNCaP cell lysateand membrane fractions, as well as in PSM-transfected PC-3 cells but notin native PC-3 cells. This detectable expression in the transfected PC-3cells proves that the previously cloned 2.65 kb PSM cDNA encodes theantigen recognized by the 7E11-C5.3 anti-prostate monoclonal antibody.

PSM mRNA Expression: Expression of PSM mRNA in normal human tissues wasanalyzed using ribonuclease protection assays. Tissue expression of PSMappears predominantly within the prostate, with very low levels ofexpression detectable in human brain and salivary gland (FIG. 20). Nodetectable PSM mRNA expression was evident in non-prostatic humantissues when analyzed by Northern analysis. On occasion it is noted thatdetectable PSM expression in normal human small intestine tissue,however this mRNA expression is variable depending upon the specificriboprobe used. All samples of normal human prostate and human prostaticadenocarcinoma assayed have revealed clearly detectable PSM expression,whereas generally decreased or absent expression of PSM in tissuesexhibiting benign hyperplasia (FIG. 21). In human LNCaP tumors grownboth orthotopically and subcutaneously in nude mice abundant PSMexpression with or without the use of matrigel, which is required forthe growth of subcutaneously implanted LNCaP cells was detected (FIG.21). PSM mRNA expression is distinctly modulated by the presence ofsteroids in physiologic doses (FIG. 22). DHT downregulated expression by8-10 fold after 24 hours and testosterone diminished PSM expression by3-4 fold. Estradiol and progesterone also downregulated PSM expressionin LNCaP cells, perhaps as a result of binding to the mutated androgenreceptor known to exist in the LNCaP cell. Overall, PSM expression ishighest in the untreated LNCaP cells grown in steroid-depleted media, asituation that simulates the hormone-deprived (castrate) state in-vivo.This experiment was repeated at steroid dosages ranging from 2-200 nMand at time points from 6 hours to 7 days with similar results; maximaldownregulation of PSM mRNA was seen with DHT at 24 hours at doses of2-20 nM.

Experimental Discussion

Previous research has provided two valuable prostatic bio-markers, PAPand PSA, both of which have had a significant impact on the diagnosis,treatment, and management of prostate malignancies. The present workdescribing the preliminary characterization of the prostate-specificmembrane antigen (PSM) reveals it to be a gene with many interestingfeatures. PSM is almost entirely prostate-specific as are PAP and PSA,and as such may enable further delineation of the unique functions andbehavior of the prostate. The predicted sequence of the PSM protein (30)and its presence in the LNCaP cell membrane as determined by Westernblotting and immunohistochemistry, indicate that it is an integralmembrane protein. Thus, PSM provides an attractive cell surface epitopefor antibody-directed diagnostic imaging and cytotoxic targetingmodalities. The ability to synthesize the PSM antigen in-vitro and toproduce tumor xenografts maintaining high levels of PSM expressionprovides us with a convenient and attractive model system to furtherstudy and characterize the regulation and modulation of PSM expression.Also, the high level of PSM expression in the LNCaP cells provides anexcellent in-vitro model system. Since PSM expression ishormonally-responsive to steroids and may be highly expressed inhormone-refractory disease. The detection of PSM mRNA expression inminute quantities in brain, salivary gland, and small intestine warrantsfurther investigation, although these tissues were negative forexpression of PSM antigen by immunohistochemistry using the 7E11-C5.3antibody. In all of these tissues, particularly small intestine, mRNAexpression using a probe corresponding to a region of the PSM cDNA nearthe 3′ end, whereas expression when using a 5′ end PSM probe was notdetected. These results may indicate that the PSM mRNA transcriptundergoes alternative splicing in different tissues.

Applicants approach is based on prostate tissue specific promotor:enzyme or cytokine chimeras. Promotor specific activation of prodrugssuch as non toxic gancyclovir which is converted to a toxic metaboliteby herpes simplex thymidine kinase or the prodrug4-(bis(2chloroethyl)amino)benzoyl-1-glutamic acid to the benzoic acidmustard alkylating agent by the pseudomonas carboxy peptidase G2 wasexamined. As these drugs are activated by the enzyme (chimera)specifically in the tumor the active drug is released only locally inthe tumor environment, destroying the surrounding tumor cells. Promotorspecific activation of cytokines such as IL-12, IL-2 or GM-CSF foractivation and specific antitumor vaccination is examined. Lastly thetissue specific promotor activation of cellular death genes may alsoprove to be useful in this area.

Gene Therapy Chimeras: The establishment of “chimeric DNA” for genetherapy requires the joining of different segments of DNA together tomake a new DNA that has characteristics of both precursor DNA speciesinvolved in the linkage. In this proposal the two pieces being linkedinvolve different functional aspects of DNA, the promotor region whichallows for the reading of the DNA for the formation of mRNA will providespecificity and the DNA sequence coding for the mRNA will provide fortherapeutic functional DNA.

DNA-Specified Enzyme or Cytokine mRNA: When effective, antitumor drugscan cause the regression of very large amounts of tumor. The mainrequirements for antitumor drug activity is the requirement to achieveboth a long enough time (t) and high enough concentration (c) (cxt) ofexposure of the tumor to the toxic drug to assure sufficient cell damagefor cell death to occur. The drug also must be “active” and the toxicityfor the tumor greater than for the hosts normal cells. The availabilityof the drug to the tumor depends on tumor blood flow and the drugsdiffusion ability. Blood flow to the tumor does not provide forselectivity as blood flow to many normal tissues is often as great orgreater than that to the tumor. The majority of chemotherapeuticcytotoxic drugs are often as toxic to normal tissue as to tumor tissue.Dividing cells are often more sensitive than non-dividing normal cells,but in many slow growing solid tumors such as prostatic cancer this doesnot provide for antitumor specificity.

Previously a means to increase tumor specificity of antitumor drugs wasto utilize tumor associated enzymes to activate nontoxic prodrugs tocytotoxic agents. A problem with this approach was that most of theenzymes found in tumors were not totally specific in their activity andsimilar substrate active enzymes or the same enzyme at only slightlylower amounts was found in other tissue and thus normal tissues werestill at risk for damage.

To provide absolute specificity and unique activity, viral, bacterialand fungal enzymes which have unique specificity for selected prodrugswere found which were not present in human or other animal cells.Attempts to utilize enzymes such as herpes simplex thymidine kinase,bacterial cytosine deaminase and carboxypeptidase G-2 were linked toantibody targeting systems with modest success. Unfortunately, antibodytargeted enzymes limit the number of enzymes available per cell. Also,most antibodies do not have a high tumor target to normal tissue ratiothus normal tissues are still exposed reducing the specificity of theseunique enzymes. Antibodies are large molecules that have poor diffusionproperties and the addition of the enzymes molecular weight furtherreduces the antibodies diffusion.

Gene therapy could produce the best desired result if it could achievethe specific expression of a protein in the tumor and not normal tissuein order that a high local concentration of the enzyme be available forthe production in the tumor environment of active drug.

Cytokines:

Results demonstrated that tumors such as the bladder and prostate werenot immunogenic, that is the administration of irradiated tumor cells tothe animal prior to subsequent administration of non-irradiated tumorcells did not result in a reduction of either the number of tumor cellsto produce a tumor nor did it reduce the growth rate of the tumor. Butif the tumor was transfected with a retrovirus and secreted largeconcentrations of cytokines such as Il-2 then this could act as anantitumor vaccine and could also reduce the growth potential of analready established and growing tumor. IL-2 was the best, GM-CSF alsohad activity whereas a number of other cytokines were much less active.In clinical studies just using IL-2 for immunostimulation, very largeconcentrations had to be given which proved to be toxic. The key to thesuccess of the cytokine gene modified tumor cell is that the cytokine isproduced at the tumor site locally and is not toxic and that itstimulates immune recognition of the tumor and allows specific and nontoxic recognition and destruction of the tumor. The exact mechanisms ofhow IL-2 production by the tumor cell activates immune recognition isnot fully understood, but one explanation is that it bypasses the needfor cytokine production by helper T cells and directly stimulates tumorantigen activated cytotoxic CD8 cells. Activation of antigen presentingcells may also occur.

Tissue Promotor-Specific Chimera DNA Activation Non-Prostatic TumorSystems:

It has been observed in non-prostatic tumors that the use of promotorspecific activation can selectively lead to tissue specific geneexpression of the transfected gene. In melanoma the use of thetyrosinase promotor which codes for the enzyme responsible for melaninexpression produced over a 50 fold greater expression of the promotordriven reporter gene expression in melanoma cells and not non melanomacells. Similar specific activation was seen in the melanoma cellstransfected when they were growing in mice. In that experiment nonon-melanoma or melanocyte cell expressed the tyrosinase drive reportergene product. The research group at Welcome Laboratories have cloned andsequenced the promoter region of the gene coding for carcinoembryonicantigen (CEA). CEA is expressed on colon and colon carcinoma cells butspecifically on metastatic. A gene chimera was generated which cytosinedeaminase. Cytosine deaminase which converts 5 fluororocytosine into 5fluorouracil and observed a large increase in the ability to selectivelykill CEA promotor driven colon tumor cells but not normal liver cells.In vivo they observed that bystander tumor cells which were nottransfected with the cytosine deaminase gene were also killed, and thatthere was no toxicity to the host animal as the large tumors wereregressing following treatment. Herpes simplex virus, (HSV), thymidinekinase similarly activates the prodrug gancyclovir to be toxic towardsdividing cancer cells and HSV thymidine kinase has been shown to bespecifically activatable by tissue specific promoters.

Prostatic Tumor Systems: The therapeutic key to effective cancer therapyis to achieve specificity and spare the patient toxicity. Gene therapymay provide a key part to specificity in that non-essential tissues suchas the prostate and prostatic tumors produce tissue specific proteins,such as acid phosphatase (PAP), prostate specific antigen (PSA), and agene which was cloned, prostate-specific membrane antigen (PSM). Tissuessuch as the prostate contain selected tissue specific transcriptionfactors which are responsible for binding to the promoter region of theDNA of these tissue specific mRNA. The promoter for PSA has been cloned.Usually patients who are being treated for metastatic prostatic cancerhave been put on androgen deprivation therapy which dramatically reducesthe expression of mRNA for PSA. PSM on the other hand increases inexpression with hormone deprivation which-means it would be even moreintensely expressed on patients being treated with hormone therapy.

Example 3 Cloning and Characterization of the Prostate Specific MembraneAntigen (PSM) Promoter

The expression and regulation of the PSM gene is complex. Byimmunostaining, PSM antigen was found to be expressed brilliantly inmetastasized tumor, and in organ confined tumor, less so in normalprostatic tissue and more heterogenous in BPH. PSM is strongly expressedin both anaplastic and hormone refractory tumors. PSM mRNA has beenshown to be down regulated by androgen. Expression of PSM RNA is alsomodulated by a host of cytokines and growth factors. Knowledge of theregulation of PSM expression should aid in such diagnostic andtherapeutic strategies as immunoscintigraphic imaging of prostate cancerand prostate-specific promoter-driven gene therapy.

Sequencing of a 3 kb genomic DNA clone revealed that two stretches ofabout 300 B.P. (−260 to −600; and −1325 to −1625) have substantialhomology (79-87%) to known genes. The promoter lacks a GC rich region,nor does it have a consensus TATA box. However, it contains a TA-richregion from position −35 to −65.

Several consensus recognition sites for general transcription factorssuch as AP1, AP2, NFkB, GRE and E2-RE were identified. Chimericconstructs containing fragments of the upstream region of the PSM genefused to a promoterless chloramphenicol acetyl transferase gene weretransfected into, and transiently expressed in LNCaP, PC-3, and SW620 (acolonic cell line). With an additional SV40 enhancer, sequence from −565to +76 exhibited promoter activity in LNCaP but not in PC-3 nor inSW620.

Materials and Methods

Cell Lines. LNCaP and PC-3 prostatic carcinoma cell lines (American TypeCulture Collection) were cultured in RPMI and MEM respectively,supplemented with 5% fetal calf serum at 37° C. and 5% CO₂. SW620, acolonic cell line.

Polymerase Chain Reaction. The reaction was performed in a 50 l volumewith a final concentration of the following reagents: 16.6 mM NH₄SO₄, 67mM Tris-HCl pH 8.8, acetylated BSA 0.2 mg/ml, 2 mM MgCl₂, 250 μM dNTPs,10 mM β-mercaptoethanol, and 1 U of the 111 Taq polymerase (BoehringerMannhiem, Calif.). A total of 25 cycles were completed with thefollowing profile: cycle 1, 94′C 4 min.; cycle 2 through 25, 94° C. 1min, 60° C. 1 min, 72° C. 1 min. The final reaction was extended for 10min at 72′C. Aliquots of the reaction were electrophoresed on 1% agarosegels in 1× Tris-acetate-EDTA buffer.

Cloning of PSM promoter. A bacteriophage P1 library of human fibroblastgenomic DNA (Genomic Systems, Inc., St. Louis, Mich.), was screenedusing a PCR method of Pierce et al. Primers located at the 5′ end of PSMcDNA were used: 5′-CTCAAAAGGGGCCGGATTTCC-3′ and5′CTCTCAATCTCACTAATGCCTC-3′. A positive clone, p683, was digested withXhoI restriction enzyme. Southern analysis of the restricted fragmentsusing a DNA probe from the extreme 5′ to the Ava-1 site of PSM cDNAconfirmed that a 3 Kb fragment contains the 5′ regulatory sequence ofthe PSM gene. The 3 kb XhoI fragment was subcloned into pKSBluescrptvectors and sequenced using the dideoxy method.

Functional Assay of PSM Promoter. Chloramphenicol Acetyl Transferase,(CAT) gene plasmids were constructed from the SmaI-HindIII fragments orsubfragements (using either restriction enzyme subfragments or PCR) byinsertion into promoterless pCAT basic or pCAT-enhancer vectors(Promega). pCAT-constructs were cotransfected with pSVβgal plasmid (5 μgof each plasmid) into cell lines in duplicates, using a calciumphosphate method (Gibco-BRL, Gaithersburg, Md.). The transfected cellswere harvested 72 hours later and assayed (15 μg of lysate) for CATactivity using the LSC method and for βgal activity (Promega). CATactivities were standardized by comparision to that of the βgalactivities.

Results Sequence of the 5′ End of the PSM Gene.

The DNA sequence of the 3 kb XhoI fragment of p683 which includes 3017by of DNA from the RNA start site was determined. (FIG. 15) The sequencefrom the XhoI fragment displayed a remarkable arrays of elements andmotifs which are characteristic of eukaryotic promoters and regulatoryregions found in other genes (FIG. 16).

Functional Analysis of Upstream PSM Genomic Elements for PromoterActivity.

Various pCAT-PSM promoter constructs were tested for promoter activitiesin two prostatic cell lines: LNCaP, PC-3 and a colonic SW620 (FIG. 17).Induction of CAT activity was neither observed in p1070-CAT whichcontained a 1070 by PSM 5′ promoter fragment, nor in p676-CAT whichcontained a 641 by PSM 5′ promoter fragment. However, with an additionalSV-40 enhancer, sequence from −641 to −1 (p676-CATE) exhibited promoteractivity in LNCaP but not in PC-3 nor in SW620.

Therefore, a LNCaP specific promoter fragment from −641 to −1 has beenisolated which can be used in PSM promoter-driven gene therapy.

Example 4 Alternatively Spliced Variants of Prostate Specific MembraneAntigen RNA: Ratio of Expression as a Potential Measurement ofProgression Materials and Methods

Cell Lines. LNCaP and PC-3 prostatic carcinoma cell lines were culturedin RPMI and MEM respectively, supplemented with 5% fetal calf serum at37° C. and 5% CO₂.

Primary tissues. Primary prostatic tissues were obtained from MSKCC'sin-house tumor procurement service. Gross specimen were pathologicallystaged by MSKCC's pathology service.

RNA Isolation. Total RNA was isolated by a modified guanidiniumthiocynate/phenol/chloroform method using a RNAzol B kit (Tel-Test,Friendswood, Tex.). RNA was stored in diethyl pyrocarbonate-treatedwater at −80° C. RNA was quantified using spectrophometric absorption at260 nm.

cDNA synthesis. Two different batches of normal prostate mRNAs obtainedfrom trauma-dead males (Clontech, Palo Alto, Calif.) were denatured at70° C. for 10 min., then reverse transcribed into cDNA using randomhexamers and Superscript II reverse transcriptase (GIBCO-BRL,Gaithersburg, Md.) at 50° C. for 30 min. followed by a 94° C. incubationfor 5 min.

Polymerase Chain Reaction. Oligonucleotide primers(5′-CTCAAAAGGGGCCGGATTTCC-3′ and 5′-AGGCTACTTCACTCAAAG-3′), specific forthe 5′ and 3′ ends of PSM cDNA were designed to span the cDNA sequence.The reaction was performed in a 50 μl volume with a final concentrationof the following reagents: 16.6 mM NH₄SO₄, 67 mM Tris-HCl pH 8.8,acetylated BSA 0.2 mg/ml, 2 mM MgCl₂, 250 μM dNTPs, 10 mMβ-mercaptoethanol, and 1 U of rTth polymerase (Perkin Elmer, Norwalk,Conn.). A total of 25 cycles were completed with the following profile:cycle 1, 94° C. 4 min.; cycle 2 through 25, 94° C. 1 min, 60° C. 1 min,72° C. 1 min. The final reaction was extended for 10 min at 72° C.Aliquots of the reaction were electrophoresed on 1% agarose gels in 1×Tris-acetate-EDTA buffer.

Cloning of PCR products. PCR products were cloned by the TA cloningmethod into pCRII vector using a kit from Invitrogen (San Diego,Calif.). Ligation mixture were transformed into competent Escherichiacoli Inv5α.

Sequencing. Sequencing was done by the dideoxy method using a sequenasekit from US Biochemical (Cleveland, Ohio). Sequencing products wereelectrophoresed on a 5% polyacrylamide/7M urea gel at 52° C.

RNase Protection Assays. Full length PSM cDNA clone was digested withNgoM 1 and NheI. A 350 b.p. fragment was isolated and subcloned intopSPORT1 vector (GIBCO-BRL, Gaithersburg, Md.). The resultant plasmid,pSP350, was linearized, and the insert was transcribed by SP6 RNApolymerase to yield antisense probe of 395 nucleotide long, of which 355nucleotides and/or 210 nucleotides should be protected from RNAsedigestion by PSM RNA respectively. Total celluar RNA (20 μg) fromdifferent tissues were hybridized to the aforementioned antisense RNAprobe. Assays were performed as described. tRNA was used as negativecontrol. RPAs for LNCaP and PC-3 were repeated.

Results

RT-PCR of mRNA from normal prostatic tissue. Two independent RT-PCR ofmRNA from normal prostates were performed as described in Materials andMethods. Subsequent cloning and sequencing of the PCR products revealedthe presence of an alternatively spliced variant, PSM′. PSM′ has ashorter cDNA (2387 nucleotides) than PSM (2653 nucleotides). The resultsof the sequence analysis are shown in FIG. 18. The cDNAs are identicalexcept for a 266 nucleotide region near the 5′ end of PSM cDNA(nucleotide 114 to 380) that is absent in PSM′ cDNA. Two independentrepetitions of RT-PCR of different mRNA samples yielded identicalresults.

RNase Protection Assays. An RNA probe complementary to PSM RNA andspanning the 3′ splice junction of PSM′ RNA was used to measure relativeexpression of PSM and PSM′ mRNAs (FIG. 19). With this probe, both PSMand PSM′ RNAs in LNCaP cells was detected and the predominant form wasPSM. Neither PSM nor PSM′ RNA was detected in PC-3 cells, in agreementwith previous Northern and Western blot data. FIG. 20 showed thepresence of both splice variants in human primary prostatic tissues. Inprimary prostatic tumor, PSM is the dominant form. In contrast, normalprostate expressed more PSM′ than PSM. BPH samples showed about equalexpression of both variants.

Tumor Index. The relative expression of PSM and PSM′ (FIG. 36) wasquantified by densitometry and expressed as a tumor index (FIG. 21).LNCaP has an index ranging from 9-11; CaP from 3-6; BPH from 0.75 to1.6; normal prostate has values from 0.075 to 0.45.

Discussion

Sequencing data of PCR products derived from human normal prostatic mRNAwith 5′ and 3′ end PSM oligonucleotide primers revealed a second splicevariant, PSM′, in addition to the previously described PSM cDNA.

PSM is a 750 a.a. protein with a calculated molecular weight of 84,330.PSM was hypothesized to be a type II integral membrane protein. Aclassic type II membrane protein is the transferrin receptor and indeedPSM has a region that has modest homology with the transferrin receptor.Analysis of the PSM amino acid sequence by either the methods of Rao andArgos or Eisenburg et. al. strongly predicted one transmembrane helix inthe region from a.a.#20 to #43. Both programs found other regions thatcould be membrane associated but were not considered likely candidatesfor being transmembrane regions.

PSM′ antigen, on the other hand, is a 693 a.a. protein as deduced fromits mRNA sequence with a molecular weight of 78,000. PSM′ antigen lacksthe first 57 amino acids present in PSM antigen (FIG. 18). It is likelythat PSM′ antigen is cytosolic.

The function of PSM and PSM′ are probably different. The cellularlocation of PSM antigen suggests that it may interact with either extra-or intra-cellular ligand(s) or both; while that of PSM′ implies thatPSM′ can only react with cytosolic ligand(s). Furthermore, PSM antigenhas 3 potential phosphorylation sites on its cytosolic domain. Thesesites are absent in PSM′ antigen. On the other hand, PSM′ antigen has 25potential phosphorylation sites, 10 N-myristoylation sites and 9N-glycosylation sites. For PSM antigen, all of these potential siteswould be on the extracellular surface. The modifications of these sitesfor these homologous proteins would be different depending on theircellular locations. Consequently, the function(s) of each form woulddepend on how they are modified.

The relative differences in expression of PSM and PSM′ by RNaseprotection assays was analyzed. Results of expression of PSM and PSM′ inprimary prostatic tissues strongly suggested a relationship between therelative expression of these variants and the status of the cell: eithernormal or cancerous. While it is noted here that the sample size of thestudy is small (FIGS. 20 and 21), the consistency of the trend isevident. The samples used were gross specimens from patients. Theresults may have been even more dramatic if specimens that were pure incontent of CaP, BPH or normal had been used. Nevertheless, in thesespecimens, it is clear that there is a relative increase of PSM overPSM′ mRNA in the change from normal to CaP. The Tumor Index (FIG. 21)could be useful in measuring the pathologic state of a given sample. Itis also possible that the change in expression of PSM over PSM′ may be areason for tumor progression. A more differentiated tumor state may berestored by PSM′ either by transfection or by the use of differentiationagents.

Example 5 Enhanced Detection of Prostatic Hematogenous Micro-Metastaseswith PSM Primers as Compared to PSA Primers Using a Sensitive NestedReverse Transcriptase-PCR Assay

77 randomly selected samples were analyzed from patients with prostatecancer and reveals that PSM and PSA primers detected circulatingprostate cells in 48 (62.3%) and 7 (9.1%) patients, respectively. Intreated stage D disease patients, PSM primers detected cells in 16 of 24(66.7%), while PSA primers detected cells in 6 of 24 patients (25%). Inhormone-refractory prostate cancer (stage D3), 6 of 7 patients werepositive with both PSA and PSM primers. All six of these patients diedwithin 2-6 months of their assay, despite aggressive cytotoxicchemotherapy, in contrast to the single patient that tested negativelyin this group and is alive 15 months after his assay, suggesting thatPSA-PCR positivity may serve as a predictor of early mortality. Inpost-radical prostatectomy patients with negative serum PSA values, PSMprimers detected metastases in 21 of 31 patients (67.7%), while PSAprimers detected cells in only 1 of 33 (3.0%), indicating thatmicrometastatic spread may be a relatively early event in prostatecancer. The analysis of 40 individuals without known prostate cancerprovides evidence that this assay is highly specific and suggests thatPSM expression may predict the development of cancer in patients withoutclinically apparent prostate cancer. Using PSM primers, micrometastaseswere detected in 4 of 40 controls, two of whom had known BPH by prostatebiopsy and were later found to have previously undetected prostatecancer following repeat prostate biopsy performed for a rising serum PSAvalue. These results show the clinical significance of detection ofhematogenous micrometastatic prostate cells using PSM primers andpotential applications of this molecular assay.

Example 6 Modulation of Prostate Specific Membrane Antigen (PSM)Expression In Vitro by Cytokines and Growth Factors

The effectiveness of CYT-356 imaging is enhanced by manipulatingexpression of PSM. PSM mRNA expression is downregulated by steroids.This is consistent with the clinical observations that PSM is stronglyexpressed in both anaplastic and hormone refractory lesions. Incontrast, PSA expression is decreased following hormone withdrawal. Inhormone refractory disease, it is believed that tumor cells may produceboth growth factors and receptors, thus establishing an autocrine loopthat permits the cells to overcome normal growth constraints. Manyprostate tumor epithelial cells express both TGFα and its receptor,epidermal growth factor receptor. Results indicate that the effects ofTGFα and other selected growth factors and cytokines on the expressionof PSM in-vitro, in the human prostatic carcinoma cell line LNCaP.

2×10⁶ LNCaP cells growing in androgen-depleted media were treated for 24to 72 hours with EGF, TGFα, TNFβ or TNFα in concentrations ranging from0.1 ng/ml to 100 ng/ml. Total RNA was extracted from the cells and PSMmRNA expression was quantitated by Northern blot analysis and laserdensitometry. Both b-FGF and TGFα yielded a dose-dependent 10-foldupregulation of PSM expression, and EGF a 5-fold upregulation, comparedto untreated LNCaP. In contrast, other groups have shown a markeddownregulation in PSA expression induced by these growth factors in thissame in-vitro model. TNFα, which is cytotoxic to LNCaP cells, and TNFβdownregulated PSM expression 8-fold in androgen depleted LNCaP cells.

TGFα is mitogenic for aggressive prostate cancer cells. There aremultiple forms of PSM and only the membrane form is found in associationwith tumor progression. The ability to manipulate PSM expression bytreatment with cytokines and growth factors may enhance the efficacy ofCytogen 356 imaging, and therapeutic targeting of prostatic metastases.

Example 7 Neoadjuvant Androgen-Deprivation Therapy (ADT) Prior toRadical Prostatectomy Results in a Significantly Decreased Incidence ofResidual Micrometastatic Disease as Detected by Nested RT-PCT withPrimers.

Radical prostatectomy for clinically localized prostate cancer isconsidered by many the “gold standard” treatment. Advances over the pastdecade have served to decrease morbidity dramatically. Improvementsintended to assist clinicians in better staging patients preoperativelyhave been developed, however the incidence of extra-prostatic spreadstill exceeds 50%, as reported in numerous studies. A phase IIIprospective randomized clinical study designed to compare the effects ofADT for 3 months in patients undergoing radical prostatectomy withsimilarly matched controls receiving surgery alone was conducted. Thepreviously completed phase II study revealed a 10% margin positive ratein the ADT group (N=69) as compared to a 33% positive rate (N=72) in thesurgery alone group.

Patients who have completed the phase III study were analyzed todetermine if there are any differences between the two groups withrespect to residual micrometastatic disease. A positive PCR result in apost-prostatectomy patient identifies viable metastatic cells in thecirculation.

Nested RT-PCR was performed with PSM primers on 12 patients from the ADTgroup and on 10 patients from the control group. Micrometastatic cellswere detected in 9/10 patients (90%) in the control group, as comparedto only 2/12 (16.7%) in the ADT group. In the ADT group, 1 of 7 patientswith organ-confined disease tested positively, as compared to 3 of 3patients in the control group. In patients with extra-prostatic disease,1 of 5 were positive in the ADT group, as compared to 6 of 7 in thecontrol group. These results indicate that a significantly higher numberof patients may be rendered tumor-free, and potentially “cured” by theuse of neoadjuvant ADT.

Example 8 Sensitive Nested RT-PCR Detection of Circulation ProstaticTumor Cells Comparison of PSM and PSA-Based Assays

Despite the improved and expanded arsenal of modalities available toclinician today, including sensitive serum PSA assays, CT scan,transrectal ultrasonography, endorectal co.I MRI, etc., many patientsare still found to have metastatic disease at the time of pelvic lymphnode dissection and radical prostatectomy. A highly sensitive reversetranscription PCR assay capable of detecting occult hematogenousmicrometastatic prostatic cells that would otherwise go undetected bypresently available staging modalities was developed. This assay is amodification of similar PCR assays performed in patients with prostatecancer and other malignancies. The assay employs PCR primers derivedfrom the cDNA sequences of prostate-specific antigen⁶ and theprostate-specific membrane antigen recently cloned and sequenced.

Materials and Methods

Cells and Reagents. LNCaP and MCF-7 cells were obtained from theAmerican Type Culture Collection (Rockville, Md.). Details regarding theestablishment and characteristics of these cell. Cells grown in RPMI1640 medium and supplemented with L-glutamine, nonessential amino acids,and 5% fetal calf serum (Gibco-BRL, Gaithersburg, Md.) In a 5% CO₂incubator at 37° C. All cell media was obtained from the MSKCC MediaPreparation Facility. Routine chemical reagents were of the highestgrade possible and were obtained from Sigma Chemical Company (St. Louis,Mo.).

Patient Blood Specimens. All blood specimens used in this study werefrom patients seen in the outpatient offices of urologists on staff atMSKCC. Two anti-coagulated tubes per patient were obtained at the timeof their regularly scheduled blood draws. Specimens were obtained withinformed consent of each patient, as per a protocol approved by theMSKCC Institutional Review Board. Samples were promptly brought to thelaboratory for immediate processing. Seventy-seven specimens frompatients with prostate cancer were randomly selected and delivered tothe laboratory “blinded” along with samples from negative controls forprocessing. These included 24 patients with stage D disease (3 with D₀,3 with D¹, 11 with D², and 7 with D³), 31 patients who had previouslyundergone radical prostatectomy and had undetectable postoperative serumPSA levels (18 with pT2 lesions, 11 with pT3, and 2 pT4), 2 patientswith locally recurrent disease following radical prostatectomy, 4patients who had received either external beam radiation therapy orinterstitial l¹²⁵ implants, 10 patients with untreated clinical stageT1-T2 disease, and 6 patients with clinical stage T3 disease onanti-androgen therapy. The forty blood specimens used as negativecontrols were from 10 health males, 9 males with biopsy-proven BPH andelevated serum PSA levels, 7 healthy females, 4 male patients with renalcell carcinoma, 2 patients with prostatic intraepithelial neoplasia(PIN), 2 patients with transitional cell carcinoma of the bladder and apathologically normal prostate, 1 patient with acute prostatitis, 1patient with acute promyelocytic leukemia, 1 patient with testicularcancer, 1 female patient with renal cell carcinoma, 1 patient with lungcancer, and 1 patient with a cyst of the testicle.

Blood Sample Processing/RNA Extraction. 4 ml of whole anticoagulatedvenous blood was mixed with 3 ml of ice cold PBS and then carefullylayered atop 8 ml of Ficoll (Pharmacia, Uppsala, Sweden) in a 14-mlpolystyrene tube. Tubes were centrifuged at 200×g for 30 min. at 4° C.The buffy coat layer (approx. 1 ml.) was carefully removed and redilutedto 50 ml with ice cold PBS in a 50 ml polypropylene tube. This tube wasthen centrifuged at 2000×g for 30 min. at 4° C. The supernatant wascarefully decanted and the pellet was allowed to drip dry. One ml ofRNazol B was then added to the pellet and total RNA was isolated as permanufacturers directions (Cinna/Biotecx, Houston, Tex.) RNAconcentrations and purity were determined by UV spectroscopy on aBeckman DU 640 spectrophotometer and by gel analysis.

Determination of PCR Sensitivity. RNA was isolated from LNCaP cells andfrom mixtures of LNCaP and MCF-7 cells at fixed ratios (i.e. 1:100,1:1,000, etc.) using RNAzol B. Nested PCR was then performed asdescribed below with both PSA and PSM primers in order to determine thelimit of detection for the assay. LNCaP:MCF-7 (1:100,000) cDNA wasdiluted with distilled water to obtain concentrations of 1:1,000,000.The human breast cancer cell line MCF-7 was chosen because they hadpreviously been tested by us and shown not to express either PSM nor PSAby both immunohistochemistry and conventional and nested PCR.

Polymerase Chain Reaction. The PSA outer primer sequences arenucleotides 494-513 (sense) in exon 4 and nucleotides 960-979(anti-sense) in exon 5 of the PSA cDNA. These primers yield a 486 by PCRproduct from PSA cDNA that can be distinguished from a productsynthesized from possible contaminating genomic DNA.

PSA-494 5′-TAC CCA CTG CAT CAG GAA CA-3′ PSA-9605′-CCT TGA AGC ACA CCA TTA CA-3′

The PSA inner upstream primer begins at nucleotide 559 and thedownstream primer at nucleotide 894 to yield a 355 by PCR product.

PSA-559 5′-ACA CAG GCC AGG TAT TTC AG-3′ PSA-8945′-GTC CAG CGT CCA GCA CAC AG-3′

All primers were synthesized by the MSKCC Microchemistry Core Facility.5 μg of total RNA was reverse-transcribed into cDNA using random hexamerprimers (Gibco-BRL) and Superscript II reverse transcriptase (Gibco-BRL)according to the manufacturers recommendations. 1 μl of this cDNA servedas the starting template for the outer primer PCR reaction. The 20 μlPCR mix included: 0.5 U Taq polymerase (Promega) Promega reactionbuffer, 1.5 mM MgCl₂, 200 μM dNTPs, and 1.0 μM of each primer. This mixwas then transferred to a Perkin Elmer 9600 DNA thermal cycler andincubated for 25 cycles. The PCR profile was as follows: 94° C.×15 sec.,60° C.×15 sec., and 72° C. for 45 sec. After 25 cycles, samples wereplaced on ice, and 1 μl of this reaction mix served as the template foranother 25 cycles using the inner primers. The first set of tubes werereturned to the thermal cycler for 25 additional cycles. The PSM outerupstream primer sequences are nucleotides 1368-1390 and the downstreamprimers are nucleotides 1995-2015, yielding a 67 by PCR product.

PSM-1368  5′-CAG ATA TGT CAT TCT GGG AGG TC-3′ PSM-2015 5′-AAC ACC ATC CCT CCT CGA ACC-3′

The PSM inner upstream primer span nucleotides 1689-1713 and thedownstream primer span nucleotides 1899-1923, yielding a 234 by PCRproduct.

PSM-1689  5′-CCT AAC AAA AGA GCT GAA AAG CCC-3′ PSM-1923 5′-ACT GTG ATA CAG TGG ATA GCC GCT-3′

2 μl of cDNA was used as the starting DNA template in the PCR assay. The50 μl PCR mix included: 1 U Taq polymerase (Boehringer Mannheim), 250 μMcNTPs, 10 mM β-mercaptoethanol, 2 mM MgCl₂, and 5 μl of a 10× buffer mixcontaining: 166 mM NH₄SO₄, 670 mM Tris pH 8.8, and 2 mg/ml of acetylatedBSA. PCR was carried out in a Perkin Elmer 480 DNA thermal cycler withthe following parameters: 94° C.×4 minutes for 1 cycle, 94° C.×30 sec.,58° C.×1 minute, and 72° C.×1 minute for 25 cycles, followed by 72°C.×10 minutes. Samples were then iced and 2.5 μl of this reaction mixwas used as the template for another 25 cycles with a new reaction mixcontaining the inner PSM primers. cDNA quality was verified byperforming control reactions using primers derived from theβ-2-microglobulin gene sequence¹⁰ a ubiquitous housekeeping gene. Theseprimers span exons 2-4 and generate a 620 by PCR product. The sequencesfor these primers are:

B-2 (exon 2) 5′-AGC AGA GAA TGG AAA GTC AAA-3′ B-2 (exon 4)5′-TGT TGA TGT TGG ATA AGA GAA-3′

The entire PSA mix and 7-10 μl of each PSM reaction mix were run on1.5-2% agarose gels, stained with ethidium bromide and photographed inan Eage Eye Video Imaging System (Statagene, Torrey Pines, Calif.).Assays were repeated at least twice to verify results.

Cloning and Sequencing of PCR Products. PCR products were cloned intothe pCR II plasmid vector using the TA cloning system (Invitrogen).These plasmids were transformed into competent E. coli cells usingstandard methods¹¹ and plasmid DNA was isolated using Magic Minipreps(Promega) and screened by restriction analysis. Double-stranded TAclones were then sequenced by the dideoxy method using ^(3S)S-cCTP (NEN)and Sequenase (U.S. Biochemical). Sequencing products were then analyzedon 6% polyacrilamide/7M urea gels, which were fixed, dried, andautoradiographed as described.

Southern Analysis. PCR products were transferred from ethidium-stainedagarose gels to Nytran nylon membranes (Schletcher and Schuell) bypressure blotting with a Posi-blotter (Stratagene) according to themanufacturer's instructions. DNA was cross-linked to the membrane usinga UV Stratalinker (Stratagene). Blots were pre-hybridized at 65° C. for2 hours and subsequently hybridized with denatured ³²P-labeled,random-primed cDNA probes (either PSA or PSM).^(6,7) Blots were washedtwice in 1×SSC/0.5% SDS at 42° C. and twice in 0.1×SSC/0.1% SDS at 50°C. for 20 minutes each. Membranes were air-dried and autoradiographedfor 1-3 hours at room temperature with Hyperfilm MP (Amersham).

Results

PSA and PSM Nested PCR Assays: The application of nested PCR increasedthe level of detection from an average of 1:10,000 using outer primersalone, to better than 1:1,000,000. Dilution curves demonstrating thisadded sensitivity are shown for PSA and PSM-PCR in FIGS. 1 and 2respectively. FIG. 1 shows that the 486 by product of the PSA outerprimer set is clearly detectable with ethidium staining to 1:10,000dilutions, whereas the PSA inner primer 355 by product is clearlydetectable in all dilutions shown. In FIG. 2 the PSM outer primer 647 byproduct is also clearly detectable in dilutions to only 1:10,000 withconventional PCR, in contrast to the PSM inner nested PCR 234 by productwhich is detected in dilutions as low as 1:1,000,000. Southern blottingwas performed on all controls and most of the patient samples in orderto confirm specificity. Southern blots of the respective dilution curvesconfirmed the primer specificities but did not reveal any significantlyincreased sensitivity.

PCR in Negative Controls: Nested PSA and PSM PCR was performed on 40samples from patients and volunteers as described in the methods andmaterials section. FIG. 48 reveals results from 4 representtive negativecontrol specimens, in addition to a positive control. Each specimen inthe study was also assayed with the β-2-midroglobulin control, as shownin the figure, in order to verify RNA integrity. Negative results wereobtained on 39 of these samples using the PSA primers, however PSMnested PCR yielded 4 positive results. Two of these “false positives”represented patients with elevated serum PSA values and an enlargedprostate who underwent a transrectal prostate biopsy revealing stromaland fibromuscular hyperplasia. In both of these patients the serum PSAlevel continued to rise and a repeat prostate biopsy performed at alater date revealed prostate cancer. One patient who presented to theclinic with a testicular cyst was noted to have a positive PSM nestedPCR result which has been unable to explain. Unfortunately, this patientnever returned for follow up, and thus have not been able to obtainanother blood sample to repeat this assay. Positive result were obtainedwith both PSA and PSM primers in a 61 year old male patient with renalcell carcinoma. This patient has a normal serum PSA level and a normaldigital rectal examination. Overall, if the two patients were excludedin whom a positive PCR, but no other clinical test, accurately predictedthe presence of prostate cancer, 36/38 (94.7%) of the negative controlswere negative with PSM primers, and 39/40 (97.5%) were negative usingPSA primers.

Patient Samples: In a “blinded” fashion, in which the laboratory staffwere unaware of the nature of each specimen, 117 samples from 77patients mixed randomly with 40 negative controls were assayed. Thepatient samples represented a diverse and heterogeneous group asdescribed earlier. Several representative patient samples are displayedin FIG. 49, corresponding to positive results from patients with bothlocalized and disseminated disease. Patients 4 and 5, both with stage Dprostate cancer exhibit positive results with both the outer and innerprimer pairs, indicating a large circulating tumor cell burden, ascompared to the other samples. Although the PSM and PSA primers yieldedsimilar sensitivities in LNCaP dilution curves as previously shown, PSMprimers detected micrometastases in 62.3% of the patient samples,whereas PSA primers only detected 9.1%. In patients with documentedmetastatic prostate cancer (stages D₀-D₃) receiving anti-androgentreatment, PSM primers detected micrometastases in 16/24 (66.7%),whereas PSA primers detected circulating cells in only 6/24 (25%). Inthe study 6/7 patients with hormone-refractory prostate cancer (stageD₃) were positive. In the study, PSA primers revealed micrometastaticcells in only 1/15 (6.7%) patients with either pT3 or pT4(locally-advanced) prostate cancer following radical prostatectomy. PSMprimers detected circulating cells in 9/15 (60%) of these patients.Interestingly, circulating cells 13/18 (72.2%) patients with pT2(organ-confined) prostate cancer following radical prostatectomy usingPSM primers was detected. None of these patient samples were positive byPSA-PCR.

Improved and more sensitive method for the detection of minimal, occultmicrometastic disease have been reported for a number of malignancies byuse of immunohistochemical methods, as well as the polymerase chainreaction. The application of PCR to detect occult hematogenousmicrometastases in prostate cancer was first described by Moreno, et al.using conventional PCR with PSA-derived primers.

When human prostate tumors and prostate cancer cells in-vitro werestudied by immunohistochemistry and mRNA analysis, PSM appeared to behighly expressed in anaplastic cells, hormone-refractory cells, and bonymetastases, in contrast to PSA. If cells capable of hematogenousmicrometastasis represent the more aggressive and poorly-differentiatedcells, they may express a higher level of PSM per cell as compared toPSA, enhancing their detectibility by RT-PCR.

Nested RT-PCR assays are both sensitive and specific. Results have beenreliably reproduced on repeated occasions. Long term testing of bothcDNA and RNA stability is presently underway. Both assays are capable ofdetecting one prostatic cell in at least one million non-prostatic cellsof similar size. This confirms the validity of the comparison of PSM vs.PSA primers. Similar levels of PSM expression in both human prostaticcancer cells in-vivo and LNCaP cells in-vitro resulted. The specificityof the PSM-PCR assay was supported by the finding that two “negativecontrol” patients with positive PSM-PCR results were both subsequentlyfound to have prostate cancer. This suggests an exciting potentialapplication for this technique for use in cancer screening. In contrastto recently published data, significant ability for PSA primers toaccurately detect micrometastatic cells in patients with pathologicallywith pathologically organ-confined prostate cancer, despite thesensitivity of the assay failed to result. Rather a surprisingly highpercentage of patients with localized prostate cancer that harbor occultcirculating prostate cells following “curative” radical prostatectomyresults which suggests that micrometastasis is an early event inprostate cancer.

The application of this powerful new modality to potentially stageand/or follow the response to therapy in patients with prostate cancercertainly merits further investigation. In comparison to moleculardetection of occult tumor cells, present clinical modalities for thedetection of prostate cancer spread appear inadequate.

Transition of prostate cancer from androgen dependent to androgenindependent state is a clinically important step which may be caused oraccompanied by genetic changes. Expression of prostate specific membraneantigen (PSM) is most intense in LNCap cells, an androgen dependentprostate carcinoma cell line: and is not detectable in PC-3 nor inDU-145 cells, which are androgen independent prostate carcinoma celllines. A microsatellite repeat of (TTTTG), (TTTG), has been found in thefirst intron of the PSM gene. Our hypothesis is that this Microsatelliterepeat could be a cis-acting element in the regulation of PSMexpression. A polymeric chain reaction amplifying this repeat was usedto look for any gene alteration in several cell lines: LNCap, PC-3,PC-3M, DU-145 as well as in 20 paired normal and early prostatic cancers(p12-4, NO). In addition, immunohistochemistry (IHC) was used to analyzePSM expression in patient samples. By IHC, no detectable expression inDU-145, PC-3, and PC-3M was found, but all tumor expressed PSM. Furthersequencing data of the microsatellite repeat confirmed no change inLNCap, and in contrast, an amplification in PC-3 and a gross deletion inDU-145. Alteration of a T segment adjacent to the microsatellite repeatwas found in one tumor sample. These results suggest that there israrely alteration in the intronic microsatellite sequence of the PSMgene in early prostate cancer. The abnormal pattern in the absence ofexpression suggest genetic instability in the more aggressive tumorlines such as the PC-3, PC-3M and DU-145 cells.

Example 9 Chromosomal Localization of Cosmid Clones 194 and 683 byFluorescence In-Situ Hybridization

PSM was initially mapped as being located on chromosome 11p11.2-p13(FIGS. 25-27). Further information from the cDNA in-situ hybridizationsexperiments demonstrated as much hybridization on the q as p arms. Muchlarger fragments of genomic DNA was obtained as cosmids and two of theseof about 60 kilobases each one going 3′ and the other 5′ bothdemonstrated binding to chromosome 11 p and q under low stringency.However under higher stringency conditions only the binding at 11q14-q21remained. This result suggests that there is another gene on 11p that isvery similar to PSM because it is so strongly binding to nearly 120kilobases of genomic DNA (FIG. 28).

Purified DNA from cosmid clones 194 and 683 was labelled with biotindUTP by nick translation. Labelled probes were combined with shearedhuman DNA and independently hybridized to normal metaphase chromosomesderived from PHA stimulated peripheral blood lymphocytes in a solutioncontaining 50% formamide, 10% dectran sulfate, and 2×SSC. Specifichybridization signals were detected by incubating the hybridized slidesin fluoresein conjugated avidin. Following signal detection the slideswere counterstained with propidium iodide and analyzed. These firstexperiments resulted in the specific labelling of a group C chromosomeon both the long and short arms. This chromosome was believed to bechromosome 11 on the basis of its size and morphology. A second set ofexperiments were performed in which a chromosome 11 centromere specificprobe was cohybridized with the cosmid clones. These experiments werecarried out in 60% formamide in an attempt to eliminate the crossreactive signal which was observed when low stringency hybridizationswere done. These experiments resulted in the specific labelling of thecentromere and the long arm of chromosome 11. Measurements of 10specifically labelled chromosomes 11 demonstrated that the cosmid clonesare located at a position which is 44% of the distance from thecentromere to the telomere of chromosome arm 11q, an area thatcorresponds to band 14q. A total of 160 metaphase cells were examinedwith 153 cells exhibiting specific labelling.

Cloning of the 5′ upstream and 3′ downstream regions of the PSM genomicDNA. A bacteriophage P1 library of human fibroblast genomic DNA (GenomicSystems, St. Louis, Mich.) was screened using the PCR method of Pierceet. al. Primer pairs located at either the 5′ or 3′ termini of PSM cDNAwere used. Positive cosmid clones were digested with restriction enzymesand confirmed by Southern analysis using probes which were constructedfrom either the 5′ or 3′ ends of PSM cDNA. Positive clone p683 containsthe 5′ region of PSM cDNA and about 60 kb upstream region. Clone −194contains the 3′ terminal of the PSM cDNA and about 60 kb downstream.

Example 10 Peptidase Enzymatic Activity

Prostate Specific Membrane Antigen has activity as a carboxypeptidaseand acts on both gamma linked or alpha linked amino acids which haveacidic amino acids such as glutamate in the carboxy terminus.

Prostate specific membrane antigen is found in high concentration in theseminal plasma. PSM antigen has enzymatic activity withN-acetylaspartylglutamate as a substrate and enzymatic action results inthe release of, N-acetylaspartate and glutamic acid. Because PSM actionwill release glutamate, and because it is well known that the seminalfluid is highly enriched in its content of glutamic acid, the action ofPSM antigen of endogenous protein/peptide substrates may be responsiblefor generating the glutamic acid present.

It is also uncertain as to the role that seminal plasma glutamic acidplays in fertility functions. It may be that interruption of PSM antigenenzymatic activity may block the generation of glutamate and couldimpact on seminal plasma glutamic acid levels and its attendantfertility functions. Thus agents which inhibit PSM antigen may prove tobe useful in attenuating male fertility.

Example 11 Ionotropicglutamate Receptors in Prostate Tissue

Prostate Specific Membrane antigen acts on N-acetylaspartylglutamic acidto release glutamate and because a homologous protein has been found inthe rat brain which acts on N-acetylyaspartylglutamate to free glutamateand N-acetylaspartate and because these amino acids are considered tofunction as neurotransmitters, the enzyme is considered to bepotentially important in modulating neurotransmitter excitatory aminoacid signalling as a neurocarboxypeptidase. This could be important inthe prostate as well, because of the neuroendocrine nature of asubpopulation of cells in the prostate which are considered to beimportant synthesizeing neuropeptide signaling molecules. PSM antigenfrom the LNCaP cell was isolated and LNCaP cells can be induced toexhibit a “neuron like” phenotype.

Excitatory neurotransmission in the central nervous system (CNS) ismediated predominantly by glutamate receptors. Two types of glutamatereceptors have been identified in the human CNS: metabotropic receptors,which serve G-protein coupled second messenger signalling systems, andtonotropic receptors, which serve as ligand gated ion channels.Ionotropic glutamate channels can increase the inward flow of ions suchas calcium ions. This can result in the subsequent stimulation of nitricoxide, and nitric oxide modulation of a number of signalling pathways.Nitric oxide has been found to be a major signalling mechanism involvedin cell growth and death, response to inflammation, smooth muscle cellcontraction etc.

Methods: Detection of glutamate receptor expression was performed usinganti-gluR2/3 and anti-gluR4 polyclonal antibodies and antibiotinimmunohistochemical techniques in paraffin-embedded human prostatetissues.

Results: Anti-gluR2/3 immunoreactivity was unique to prostatic stromaand was absent in the prostatic epithelial compartment. Stronganti-gluR4 immunoreactivity was observed in the basal cells of theprostate. This implied a differential location and function of glutamatereceptors as defined by these antibodies.

Discussion: Distribution of glutamate receptors in the prostate has notbeen described. Basal cells are considered the precursor cell for theprostatic acinar and neuroendocrine cells of the prostate. Glutamatereceptors may provide signalling functions in their interactions withthe prostate stroma and acinar cells, and PSM may be involved in thatinteraction. Thus inhibition or enhancement of PSM activity could serveto modulate activity of the basal cells and prove to be a valuable aidfor controlling basal cell function in the prostate.

The finding of glutamate like receptors in the stroma is of interestbecause a large part of the prostate volume is due to stromal cells.Current observation have suggested that these stromal cells have asmooth muscle cell phenotype and thus the presence of glutamatereceptors may play a role in their biologic function and regulation ofdifferentiation. A most common disease in men is the abnormal benigngrowth of the prostate termed benign prostatic hyperplasia, BPH.

In areas of BPH a decrease in the level of expression of PSM antigen wasobserved. If PSM antigen activity is providing an aspect of thesignalling for normal stromal function then the abnormal growth seen inBPH may be a response to that decreased activity and agents to restoreits function could play a role in the treatment or prevention of BPH.

Altering PSM antigen function may have beneficial actions outside theprostate. In the rat CNS a protein homology to PSM antigen wasdiscovered and provides a rational to consider prostate specificmembrane antigen as a neurocarboxypeptidase. Alterations in its functionmay occur in neurotoxic disorders such as epilepsy, or ALS, alzheimers,and multiple sclerosis.

Example 12 Identification of a Membrane-Bound PteroylpolygammaglutamylCarboxypeptidase (Folate Hydrolase) that is Expressed in Human ProstaticCarcinoma

As described PSM functions as a carboxypeptidase to hydrolyze both alphaand gamma peptide linkages with amino acids such as glutamate in theterminal carboxy position. The proximal small intestine (duodenum-strongexpression PSM) but not the distal small intestine (ileum-absent PSM)was also very rich in expression of message for prostate specificmembrane antigen in RNase protection assays. PSM antigen byimmunohistochemistry was observed in the brush border membranes of theduodenum. This location was consistent with a hydrolase known as folateconjugase (folate hydrolase as a carboxypeptidase, not an endopeptidase)that had been described in the older literature, with the protein havingbeen partially purified from the human small intestine. No cloning orsequencing of this gene had been done. There is a form of folatehydrolase that is found in all cells in the lysosomes and it wasrecently sequenced. There is no sequence relationship between thelysosomal endopeptidase. Membrane fraction of the LNCaP cells was veryrich in folate hydrolase activity. The PSM specific monoclonal could beused to immunoprecipitate the folate hydrolase activity. This resultalways has the possibility that the folate hydrolase activity is not thesame as PSM antigen but is a coprecipitating contaminant. Therefore PSMantigen was transfected into PC-3 cells. PC-3 cells do not express PSMnor do they have membrane folate hydrolase activity. In cellstransfected with PSM antigen however expression of folate hydrolaseactivity was observed in the membranes. Thus PSM is a novel folatehydrolase, folate carboxypeptidase, and is active in sequentiallyremoving the terminal gamma-linked glutamates. In the proximal smallintestine it is understandable why this enzyme would be in such a place,as the majority of folate available from food is polygammglutamated andthis enzyme is responsible for its hydrolysis.

Materials: Methotrexate triglutamate (4-NH₂-10-CH₃-PteGlu₄ (MTXglu₃)),pteroylpentaglutamate (PteGlu₅), and para-aminobenzoylpentaglutamate,(pABAGlu₅) were purchased from Dr. B. Schircks Laboratories (Jona,Switzerland) and samples were >98% pure when evaluated by HPLC.N-acetyl-α-aspartylglutamate (NAAG) (40 Ci/mmol) was purchased from NewEngland Nuclear (Boston, Mass.). Protein A Sepharose 4 Fast Flow waspurchased from Pharmacia (Piscataway, N.J.). The 7E11-C5 monoclonalantibody to prostate specific membrane antigen was obtained from CytogenCorporation, Princeton, N.J. All other reagents(p-hydroxymercuribenzoate, homocysteine, dithiothreitol (DTT), reducedglutathione) were of the highest purity commercially available fromSigma Chemical Co. (St. Louis, Mo.).

Culture and growth of human prostate adenocarcinoma cells (LNCaP, PC-3,TSU-Pr1, and Duke-145): LNCaP cells were maintained in defined culturemedium, RPMI-1640 medium supplemented with non-essential amino acids, 5mM glutamine, and 5% heat-inactivated fetal calf serum. Duke-145, PC-3,and TSU-Pr1 cells were grown in minimal essential medium (MEM), Ham'sF-12K, and MEM, respectively, containing 5% fetal calf serum. Noantibiotic was included in the media. Cells (1×10⁶) were plated in T-75tissue culture flasks containing 15 mL of medium and incubated at 37° C.in a humidified atmosphere of 5% CO₂. Cell numbers were determined usinga Model Z F Coulter Counter (Coulter Electronic, Inc.). Prostate cellswere harvested from plates by gentle scraping at 4° C. into phosphatebuffered saline (136.9 mM NaCl, 2.68 mM KCl, 8.10 mM Na₂HPO₄, 1.47 mMKH₂PO₄, pH 7.34, PBS) and centrifuged at 500×g to obtain a cell pellet.Sedimented cells were routinely rinsed twice with 15 mL volumes of PBS.

Transfection of PSM into PC-3 Prostate Cell Line: The full length 2.65kb PSM cDNA was subcloned into a pREP7 eukaryotic expression vector(Invitrogen, San Diego, Calif.) as previously described. Plasmid DNA waspurified from transfected DH5-α (Gibco-BRL) using a Qiagen maxi prepplasmid isolation kit (Qiagen Inc., Chatsworth, Calif.). Purifiedplasmid DNA (5 μg) was diluted with 300 μL of serum free RPMI media andmixed with 45 μL of lipofectamine (Gibco-BRL) which was previouslydiluted with 300 μL of serum free RPMI media to allow an DNA-liposomecomplex to form. The mixture was kept at room temperature for 30minutes, then added to a 60 mm petri dish containing 60-70% confluentPC-3 cells in 2.4 mL serum free RPMI. The DNA-liposome complexcontaining serum free media was mixed gently to ensure uniformdistribution and was then incubated for 6 h at 37° C. in a CO₂incubator. Following incubation, the media containing liposome-DNAcomplex was aspirated and replaced with 6 mL of regular growth media(10% fetal bovine serum, 1% penicillin-streptomycin, 1% glutamine).After 48 hours, cells were trypsinized and split 1:3 into 60 mm dishescontaining regular media supplemented with 200 μg/mL of hygromycin B(Calbiochem, LaJolla, Calif.). Cells were maintained for 2 weeks withchanges of media containing hygromycin B every third day until discretecolonies appeared. Colonies were isolated using a 6 mm cloning cylinderand were expanded in the same media. As a control, PC-3 cells were alsotransfected with the pREP7 vector alone.

Immunohistochemistry: The 7E11-C5 monoclonal antibody to prostatespecific antigen was used. This antibody recognizes a portion ofcarbohydrate-containing peptide epitope on the amino terminal end of PSMthat is located on the inner portion of the cytosolic membrane. Afterpermeabilization of LNCaP and PC-3 transfected and non-transfected cellswith a mixture of acetone and methanol (1:1 v/v) and blocking with 5%bovine serum albumin in 50 mM Tris buffered saline (TBS) pH 7.45,samples were incubated with 7E11-C5 antibody (20 μg/mL) for 1 h at roomtemperature. Negative controls were generated by substituting the sameconcentration of mouse IgG2ak for the PSM antibody. Using a secondaryIgG₁ anti-mouse antibody conjugated with alkaline phosphatase, sampleswere re-incubated for 1 h, rinsed in TBS, and stained withbromochloroindolylphenol phosphate in 2-amino-2-methyl-1-propanolbuffer. Cells expressing PSM demonstrate an intense blue staining.

Cell Membrane Preparation: Cell lysates were prepared by sonicatingapproximately 6×10⁶ cells in 50 mM Tris pH 7.4 buffer (2×10 s pulses at20 mWatts) in an ice-bath. Membrane fractions were obtained bycentrifuging lysates at 100,000×g for 30 mins. The supernatant fractionswere saved and pelleted membranes were re-suspended by gentletrituration and re-sedimented at 100,000×g for 30 mins through 10 mL ofcold 50 mM Tris pH 7.4 buffer. Washed membrane fractions were dissolvedin 50 mM Tris pH 7.4 buffer containing 0.1% Triton X-100 (Tris/Triton).Enzymatic activity and immunoprecipitation preparations were performedusing this membrane preparation.

Immunoprecipitation of PSM from Membrane: Membrane pellets (˜1 mgprotein) solubilized in Tris/Triton buffer were incubated at 4° C. for 1h in the presence of 7E11-C5 anti-prostate monoclonal antibody (6 ugprotein). Protein A Sepharose gel equilibrated in Tris/Triton buffer wasadded to the immunocomplex. This preparation was subsequently incubatedfor an additional hour at 4° C. Sepharose beads were centrifuged at500×g for 5 mins and rinsed twice with Tris/Triton buffer at pH 7.4.Isolated beads were resuspended in 0.1 M glycine buffer pH 3.0,vortexed, and the supernatant fraction was assayed for hydrolaseactivity using MTXglu₃.

Pteroyl Gamma-Glutamyl Hydrolase Assay: Hydrolase activity wasdetermined using capillary electrophoresis. The standard assay mixturecontained 50 uM MTXGlu₃, 50 mM acetate buffer (pH 4.5) and enzyme to afinal volume of 100 uL. A sample preparation without enzyme wasincubated concurrently with enzymatic assays and reactions wereconducted for times varying between 0 and 240 min at 37° C. Activitieswere also determined in standard reaction mixture at varied pHs for 60min. Reactions were terminated in a boiling water bath for 5 min andsamples were stored frozen (−20° C.) until analysis. Followingcentrifugation (7,000×g) to remove precipitated debris, capillaryseparation of MTX glutamated analogues was performed with a SpectraPhoresis 1000 instrument (Thermo Separation, San Jose, Calif.) with a 75μm id×50 cm silica capillary (Polymicro Technology, Phoenix, Ariz.).Separation of pteroyl(glutamate)_(n) derivatives is achieved with anelectrolyte of 20 mM sodium borate with 15 mM sodium dodecylsulfate (pH9.5) with +20 Kev at 25° C. Samples were applied hydrodynamically for1-2 s and absorbance monitored at 300 nm. Data were recorded with an IBMcomputer using CE-1000 software (Thermo Separation).

Protein determination: Protein concentrations of isolated membrane orsupernatant fractions were determined by incubating diluted aliquotswith BCA reagent (Pierce Chemical Co., Rockford, Ill.) at 37° C. for 30min. The spectrophotometric quantitation of protein was conducted bydetermining the absorbance at 562 nm against bovine serum albuminstandard.

Statistical Analysis: Data were analyzed by using the Statgraphicsversion 4.0 program (Statistical graphics Corporation, Rockville, Md.)and where summarized are expressed as mean±S.D. Student's unpaired ttest was used to determine significance of differences.

Results:

Membrane fractions isolated from human prostate adenocarcinoma cells(LNCaP) were incubated using primarily MTXglu₃ as substrate. The timecourse of hydrolysis of the gamma-linked triglutamate derivatie and thesubsequent appearance of MTXglu₂, MTXglu₁, and MTX after 30, 60, 120,and 240 min of incubation are illustrated in FIG. 82. The semipurifiedPSM antigen exhibits pteroyl poly gamma-glutamyl exopeptidase activitythat progressively liberates all of the possible glutamates from MTXGlu₃with accumulation of MTX.

The PSM antigen was immunoprecipitated in the presence of 7E11-C5anti-prostate monoclonal antibody and the PSM antigen-antibody complexwas adsorbed onto a Protein A Sepharose Gel column. Following twicewashing of the sepharose beads with 2 mL volumes of buffer andre-solubilization of the antigen-antibody complex by adjusting theelution pH to 3.0, the supernatant fraction was assayed for hydrolaseactivity. FIG. 55 shows the capillary electrophoretic separation ofsuccessively cleaved glutamyl moieties from MTXglu₃ after 0, 30, 60 and240 min incubations. Results similar to these in FIG. 82 were obtainedusing pteglu₅ with formation of folate (pteglu₁).

The optimum pH activity profiles of the immunoprecipitated PSM hydrolasefrom LNCaP cells and of the membrane fractions from PC-3 PSM-transfectedand non-transfected (vector alone) cells are shown in FIG. 57. Thereaction was monitored as a function of pH from 2 to 10 after an 1 hincubation with MTXglu₃. The extent of reaction was expressed as theconcentration of MTXglu₂ formed per mg protein. Although all reactionproducts were detectable as illustrated in FIG. 56, MTXglu₂ was thepredominant hydrolyzed species at incubation times ranging from 10 to 60min. The pH profile of membrane fractions isolated from both LNCaP andPC-3 PSM-transfected cells are identical and exhibit two maxima of PSMhydrolase activity at pH 5 and 8 with no measurable activity above pH10.

To determine whether non-PSM expressing human adenocarcinoma cell lines(PC-3, TSU-Pr1, and Duke-145) exhibit folate hydrolase activity,isolated membrane preparations from these cell lines were analyzed (FIG.83). The less differentiated, hormone refractory prostate cell lines(PC-3, TSU-Pr1, and Duke-145) exhibit no appreciable activity after 2 hincubations. These results are in agreement with previous findings thatdemonstrate neither a presence of a mRNA for PSM nor antigenimmunoreactivity with 7E11-C5 in these cells.

In further studies in which the cDNA for PSM was transfected intonon-PSM antigen expressing PC-3 cells, a close correlation between PSMantigen immunoreactivity and hydrolase activity was observed withMTXglu₃ in membranes of LNCaP and PC-3 PSM-transfected cells (FIGS. 58and 59). Immunohistochemical analyses of LNCaP (FIG. 58) and PSM antigenexpressing PC-3 (FIG. 85B) cells revealed distinct positive stainingwith 7E11-C5 anti-prostate monoclonal antibody. FIG. 85C illustrates noimmunoreactivity in PC-3 cells expressing the pREP7 hygromycin vectoralone. In preparations of negative controls, all three cell lines werereacted with IgG2aK rather than with 7E11-C5 antibody. No backgroundstaining resulted with the secondary antibody conjugated with alkalinephosphatase.

To compare PSM hydrolase activity with that of other gamma-glutamylhydrolases that either reside within the lysosome or are secreted asobserved in several neoplastic cells, its reactivity in the presence ofthiol-containing reducing agents, namely, reduced glutathione,homocysteine, and dithiothreitol (DTT), and the thiol reagent,p-hydroxymercuribenzoate (PHMB), at concentrations ranging from 0.05-0.5mM was observed. Of the reduced sulfhydryl derivatives, it wasdiscovered that only DTT (≧0.2 mM) was slightly inhibitory (86±3% ofcontrol). Unlike gamma-linked peptide hydrolase retained within thelysosome, PSM hydrolase activity was maintained in the presence of 0.5mM PHMB.

The reactivity of PSM hydrolase against an α-glutamate dipeptide,N-acetyl-α-aspartylglutamate (NAAG), has been investigated and that thePSM enzyme from either LNCaP or PSM transfected PC-3 cell membraneshydrolyses NAAG producing N-acetylaspartate and glutamate was observed.Furthermore, MTXglu₃, pteglu₅, and pABAglu₅ were potent inhibitors ofthe PSM-mediated NAAG hydrolysis.

Discussion:

Membrane-bound PSM antigen has pteroyl poly gamma-glutamylcarboxypeptidase (folate hydrolase) activity. Gamma-glutamyl hydrolaseactivity is also present in lysosomes of cells and these enzymes may beresponsible for regulating the length of exogenous and endogenous folylpolyglutamate chain lengths. A characteristic difference between thesetwo hydrolases is that the PSM enzyme exhibits substantial activity atpH values 7.5 to 8.0 in addition to having an acidic pH 4.5 to 5optimum. Moderate levels of hydrolase activity are present within LNCaPcytosolic compartment and may represent the short intracellular fragmentof this class II enzyme. This reflects an interesting situation in thesecells where the majority of RNA codes for the membrane-bound enzyme thatis localized extracellularly. The ratio of the mRNAs in these samplesthat code for the class II membrane and the cytosolic proteins is ten toone. In normal prostate tissue, the mRNA coding for the membrane proteinis only one-tenth that of the cytosolic form.

It is clear from this study that the prostate specific membrane antigenfunctions as a folate hydrolase and is unique in that it has activity onboth the gamma-linked as well as the alpha linked peptide bonds. This isinteresting for a number of reasons. First in the normal prostate it wasdemonstrated that the majority of the mRNA encodes a protein, PSM′, thatis likely to be cytosolic and would imply that it may be that in theprostate that folates could exists in the lesser glutamated species. Ifso then it means that the folate in the prostate can readily leak outand that the prostate may be subjected to “microenviromental folatedeficiencies” This may be related to the high worldwide incidence of“microscopic prostate cancer” as folate deficiencies are associated withcarcinogenesis in a number of tissues.

Benign enlargement of the prostate and prostate cancer occur in oldermen. It also occurs that the uptake of folate decreases with aging. Iffolate uptake decreases with aging this may be due to decreased PSMfolate hydrolase activity in the proximal intestine. To correct such adeficiency it might be possible to use PSM folate hydrolase in foods torelease the folate before consumption or take it with foods as is donewith lactase in lactose intolerant individuals. If the prostate in menis susceptible to folate depletion then nutritional supplementation mayhelp reduce the development of the microscopic lesion, indeed in somecancers such as cancer of the colon, folate supplementation was found toreduce cancer formation.

Why would the prostate cells prefer to have the lesser glutamated formsof folate? It may be that methionine synthase which is an enzyme key tofolate uptake and folate utilization for one carbon methyl transfermetabolism may utilize the nonglutamated folate preferentially. Inaddition to folate deficiency, choline and methionine deficiency is alsoassociated with tumor development. If shown to modulate one carbontransfers, it might be useful to inhibit this enzyme as a means toinhibit cancer development and thus serve as a chemopreventative agent.Again modulation of PSM folate hydrolase may play a role in tumorprevention and modulation of tumor growth.

A feature that cell biologists use in transfecting DNA into cells oftenrequires selection of the transfected gene and often multipletransfections are performed. These are done with drugs that are toxic tocells such as Hygromycin and use genes that code for Hygromycinresistance which are bacterial. It may be that PSM could be used as aselectable marker by growing the transfected cells in folate free mediaand including polyglutamated folate which would be able to rescue cellsfrom folate deficiency if they expressed PSM.

PSM folate hydrolase activity can possibly be used as a prodrugconverting enzyme. In the normal prostate PSM is intracellular. In thetransformed cell the majority of the protein and its attendant enzymaticactivity is extracellular in location. It may be that as the enzymesassociated with cell growth require the polyglutamated forms the cancerfinds a way to remove PSM folate hydrolase from the interior byalternative splicing to an extracellular enzyme. PSM is a membraneprotein and is found to predominate in cancer, but PSM′ is likely acytosolic protein which predominates in the normal condition.

This implies that development of a prodrug that requires metabolismbefore it can be taken up by the tumor cell could be activated by thePSM folate hydrolase which is predominate in the cancer.

Methotrexate triglutamate was one of the agents used to identify theenzymatic activity of PSM antigen. Methotrexate triglutamate would notbe able to use the transport protein to be taken into tumor cells,because there are specific structural requirements for folate, ormethotrexate transport. If one removes the gamma-linked glutamates thenmethotrexate can be taken into cells and can exerts its antifolate,antitumor growth action.

Therfore methotrexategammatriglutamate was used to examine the action ofthis compound on the in vitro growth of PC-3 cells transfected with aplasmid with a selectable marker versus a plasmid with a selectablemarker that expresses PSM antigen as well. the PC-3 cells that weretransfected with PSM were inhibited 85% in growth by day four by 10 uMmethotrexate triglutamate, while the PC-3 plasmid only transfectants didnot exhibit any significant inhibition of growth.

PSM's folate hydrolase activity hydrolyses down to the last glutamatewhich is in alpha linked position but does not remove it. Because itdoes not remove the last glutamate, PSM antigen's folate hydrolaseactivity better serves the prodrug activation requirements of such aprodrug. Also because it is a human enzyme it is less likely than thecarboxypeptidase G2 will cause an immune response because PSM antigen isnormally present in the body.

In addition PSM could also be used as part of a prodrug strategy thatutilized gene transfer and a tissue or tumor specific promoter, say suchthat it would be linked to CEA promoter and PSM expressed in colontumors and the patients subsequently given the prodrug such asmethotrexate triglutamate. The same is also true for the protein itself,either the whole protein or the components of the active site or amodified version that would have increased prodrug activating activitycould be linked to a delivery vehicle such as an antibody or otherspecific targeting ligand, delivered to the tumor for localization andsubsequent activation.

Methotrexate as a prodrug may be enhanced in specificity by using alphalinked glutamates rather than gamma linked glutamates because theubiquitous lysosomal hydrolase enzyme is specific for the gamma linkedbond. A pro-drug with all alpha linked glutamates would not be asubstrate, but would be a substrate for the PSM folate hydrolase.

In addition to methotrexate a number of potential enzyme substrates canbe employed as cytotoxic prodrugs. The synthesis of potential prodrugs,PALAglu, and a number of other potential agents are described.

Alpha-linked methotrexate material is synthesized by the followingMerifield solid phase scheme (see FIG. 88). The scheme is based on amodification of the standard Merifield solid peptide synthesis that wasapplied to the synthesis of methotrexate y polyglutamates. In brief theN-Fmoc-4-terbutylglutamate is first connected to the resin understandard coupling conditions using diisoprpylazodicarboxylate as acoupling reagent. The Fmoc protecting group is then removed withpiperidine, and this cycle would be reiterated for as many times asglutamates would be needed to obtain the desired analog. For example saythe pentaglutamate on solid support is the intermediate required for thepreparation of methotrexate-alpha-tetraglutamate. It is deprotected atthe terminal nitrogen by treatment with piperidine, then coupled withpteroic acid analogue under the same conditions used above. The terbutyland the resin are all removed in one step with 95% trifluoroacetic acid(TPA) to provide the desired material. This process is applied to everyanalog. The gamma linked material is provided in a similar manner foruse comparative studies with the alpha-linked material (see FIG. 89).Because of the carboxypeptidase activity a number of combination ofalpha and gamma linked acidic amino acid can be optimized for theirutilization of the enzyme and for in vivo activity. In addition to thefolate like antagonists, a number of amino acid analogs were found inthe past to have antitumor activity but lacked in vivo specificity.These agents are targetable by attaching a glutamate to the carboxyterminus of the amino acid as described and shown in the figures.

PALA-Glutamate 3 and analog 5, was synthesized in a similar manner withthe addition to the introduction of a protected phosphonoacetate moietyinstead of a simple acetate. It is compatible with the function ofdiethylphosphonoacetic acid which allows the removal of the ethyl groupsunder relatively mild conditions.

Commercially available diethylphosphonoacetic acid was treated withperfluorophenyl acetate in pyridine at 0 deg.C to room temperature foran hour to afford the corresponding pentafluorophenyl ester in nearlyquantitative yield after short path column chromatography. This was thenreacted with gamma-benzylaspartate and HOAT in tetrahydrofuran for halfan hour at reflux temperature to give protected PALA 7(N-phosphonoacetylaspartate) in 90% yield after flash columnchromatography. The free acid was then activated as itspentafluorophenyl ester 8, then it was reacted withdelta-benzyl-L-glutamate and HOAT in a mixture of THF-DMF (9:1, v/v) for12 hours at reflux to give fully protected PALA-Glutamate 9 in 66% yieldafter column chromatography. Sequential removal of the ethyl groupsfollowed by the debenzylation was accomplished for a one stepdeprotection of both the benzyl and ethyl groups. Hence protectedPALA-Glutamate was heated up to reflux in neat trimethylsilylchloridefor an overnight period. The resulting bistrimethylsilylphosphonateester 10 was submitted without purification to hydrogenolysis (H₂, 30psi, 10% Pd/C, ethylacetate). The desired material 3 was isolated afterpurification by reverse phase column chromatography and ion exchangeresin.

Analogs 4 and 5 were synthesized by preparation of phosphonoglutamate 14from the alpha-carboxyl-protected glutamate.

Commercially available alpha-benzyl-N-Boc-L-glutamate 11 was treated atrefluxing THF with neat boranedimethylsulfide complex to afford thecorresponding alcohol in 90% yield. This was transformed into bromide 12by the usual procedure (Pph₃, CBr₄).

The Michaelis-Arbuzov reaction using triethylphosphite to give thecorresponding diethylphosphonate 13 which would be deprotected at thenitrogen with trifluoroacetic acid to give free amine 14. The latterwould be condensed separately with either pentafluorophenylesters 6 or 8to give 16 and 15 respectively, under conditions similar to thosedescribed for 3. 15 and 16 would be deprotected in the same manner asfor 3 to yield desired analogs 4 and 5.

An inhibitor of the metabolism of purines and pyrimidine like DON(6-diazo-5-oxo-norleucine) or its aspartate-like 17, and glutamate-like18 analogs would be added to the series of substrates.

Analog 20 is transformed into compound 17 by treatment with oxalylchloride followed by diazomethane and deprotection under knownconditions to afford the desired analogs. In addition, azotomycin isactive only after in vivo conversion to DON which will be released afteraction of PSM on analogs 17, 18, and 19.

Representative compounds, 21 and 22, were designed based on some of thespecific effects and properties of PSM, and the unique features of somenewly discovered cytotoxic molecules with now known mode of action. Thelatter, referred to commonly as enediynes, like dynemycin A 23 and orits active analogs. The recent isolation of new natural products likeDynemycin A 23, has generated a tremendous and rapidly growing interestin the medical and chemical sciences. They have displayed cytotoxicitiesto many cancer cell lines at the sub-nanomolar level. One problem isthey are very toxic, unstable, and non-selective. Although they havebeen demonstrated, in vitro, to exert their activity through DNA damageby a radical mechanism as described below, their high level of toxicitymight imply that they should be able to equally damage anything in theirpath, from proteins to enzymes.

These molecules possess unusual structural features that provide themwith exceptional reactivities. Dynemycin A 23 is relatively stable untilthe anthraquinone moiety is bioreduced into hydroanthraquinone 24. Thistriggers a chain of events by which a diradical species 25 is generatedas a result of a Bergman cycloaromatization^(F). Diradical species 25 isthe ultimate damaging edge of dynemycin A. It subtracts 2(two) protonsfrom any neighboring molecule or molecules (ie. DNA) producing radicalstherein. These radicals in turn combine with molecular oxygen to givehydroperoxide intermediates that, in the case of DNA, lead to single anddouble strand incision, and consequent cell death. Another interestingfeature was provided by the extensive work of many organic chemists whonot only achieved the total synthesis of (+)-dynemycin A 23 and otherenediynes. but also designed and efficiently prepared simpler yet asactive analogs like 26.

Enediyne 26 is also triggerable and acts by virtue of the same mechanismas for 23. This aspect is very relevant to the present proposed study inthat 27 (a very close analog of 26) is connected to NAAG such that theNAAG-27 molecule, 21, would be inert anywhere in the body (blood,organs, normal prostate cells) except in the vicinity of prostatecancer, and metastatic cells. In this connection NAAG plays a multiplerole:

-   -   Solubilization and transport: analogs of 26-type are hydrophobic        and insoluble in aqueous media, but with a water soluble        dipeptide that is indigenous to the body, substrate 21 should        follow the ways by which NAAG is transported and stored in the        body.    -   Recognition, guidance, and selectivity: Homologs of PSM are        located in the small intestines and in the brain.

In the latter, a compound like 27 when attached to a multiply chargeddipeptide like NAAG, has no chance of crossing the blood brain barrier.In the former case, PSM homolog concentration in the small intestines isin the brush border and not likely to be exposed to prodrugs in theserum. In addition, one could enhance the selectivity of delivery of theprodrug by local injection in the prostate.

26 and its analogs are established active molecules that portray theactivity of dynemycin A. Their syntheses are described in theliterature. The total synthesis of optically active 27 has beendescribed. The synthetic scheme that for the preparation of 28 is almostthe same as that of 27. However, they differ only at the position of themethoxy group which is meta to the nitrogen in the case of 28. Thisrequires an intermediate of type 29 prepared by modification of theMyers' method.

Since NAAG is optically pure, its combination with racemic materialsometimes complicates purification of intermediates. In addition, to beable to modify the components of this system one at a time, opticallypure intermediates of the type 21 and 22 are prepared. 27 was preparedin 17 steps starting from commercially available material. Anotherinteresting feature of 27 is demonstrated in a very close analog 26, itpossesses two (2) triggers as shown by the arrows.

The oxygen and the nitrogen can both engender the Bergmancycloaromatization and hence the desired damage. The simple protectiondeprotection manipulation of either functionality should permit theselective positioning of NAAG at the nitrogen or at the oxygen centers.PSM should recognize the NAAG portion of 21 or 22, then it would removethe glutamic acid moiety. This leaves 27 attached to N-acetylaspartate.

Intramolecular assisted hydrolysis of systems like N-acetylaspartyle iswell documented in the literature. The aminoacid portion shouldfacilitate the hydrolysis of such a linkage. In the event this would notwork when NAAG is placed on the nitrogen, an alternative would be toattach NAAG to the oxygen giving rise to phenolic ester 22 which is perse labile and removable under milder conditions. PSM specific substratescan be designed that could activate pro-drugs at the site of prostatictumor cells to kill those cells.

Example 13 Genomic Organization of PSM Exon/Intron Junction Sequences

RNA is synthesized and then processed by having variable numbers ofvariable sized fragments cut out and remain in the nucleus (introns) andthe remaining fragments (exons) joined together and transported out ofthe nucleus (mRNA) for use in translation into protein in the cytoplasm.This mRNA is what make the unique protein products of the cell, proteinsof specialized cells are often made in a great abundance as are theirrespective coding mRNA's. These tissue specific mRNA's can be reversetranscribed (RT) into DNA by reverse transcriptase and amplified fordetection by polymerase chain reaction (PCR) technology and thus thetechnique is called RT-PCR. If DNA is a contaminant of the MBNA fractionit would contain the message even though it was not being transcribed.

Knowledge of the intron exon junctions allows for the selection ofprimer pairs that cross an intron junction and thus allow thedetermination of DNA contamination of the RNA preparation, if present.If the intron junction were large it would be unlikely to be amplifiedwith primers, while if the intron junction were small it would stillproduce a fragment that would be much larger than the predicted fragmentsize which is based on the cDNA sequence. Thus knowledge of theintron/exon junctions provides a control to determine if the RT-PCRproduct is contaminated with DNA. Another form of DNA that could also beamplified undesirably if present as a contaminant are pseudo genes,which are intronless forms of the mRNA that reside as DNA but are notexpressed as RNA. Thus, optimized primers for detection of PSM mRNA insamples would preferably contain sequences hybridizing across theintro/exon junctions which are as follows:

          EXON 1                 Intron 1 1F. strand CGGCTTCCTCTTCGGcggcttcctcttcgg        taggggggcgcctcgcggag . . . tatttttca1R. strand                              . . . ataaaaagtCACCAAA          Exon 2                 Intron 2 2F. strand ACATCAAGAAGTTCTacatcaagaagttct         caagtaagtccatactcgaag . . .2R. strand                         . . . caagtggtcATATATTAAAATG          Exon 3                 Intron 3 3F. strand GAAGATGGAAATGAGgaagatggaaatgag         gtaaaatataaataaataaataa...3R.                                     . . . TAAAAGTTGTGTAGT          Exon 4                 Intron 4 4F. strand AAGGAATGCCAGAGGaaggaatgccagagg         taaaaacacagtgcaacaaa...4R. strand                           . . . agagttgCCGCTAGATCACA          Exon 5                 Intron 5 5F. strand CAGAGGAAATAAGGTcagaggaaataaggt         aggtaaaaattatctctttttt . . .5R. strand                           . . . gtgttttctATTTTTACGGGT          Exon 6                 Intron 6 6F. strand GTTACCCAGCAAATGgttacccagcaatg          gtgaatgatcaatccttgaat . . .6R. strand                        . . . aaaaaaagtTTATACGAATA          Exon 7                 Intron 7 7F. strand ACAGAAGCTCCTAGAacagaagctcctaga         gtaagtttgtaagaaaccargg . . .7R. strand                       . . . aaacacaggttatcTTTTTACCCA          Exon 8                 Intron 8 8F. strand AAACTTTTCTACACAaaacttttctacaca         gttaagagactatataaatttta . . .8R. strand                      . . . aaacgtaatcaTTTTCAGTTCTAC          Exon 9                 Intron 9 9F. strand AGCAGTGGAACCAGagcagtggaaccag         gtaaaggaatcgtttgctagca . . .9R. strand                              . . . aaagaTGTCTATACAGTAA          Exon 10                Intron 10 10F. Strand CTGAAAAAGGAAGGctgaaaaaggaagg         taatacaaacaaatagcaagaa . . .          Exon 11                Intron 11 11F. Strand TGAGTGGGCAGAGG         agagg         ttagttggtaatttgctataatata . . .          Exon 12                Intron 12 12F. strand ATCTATAGAAGGgtagtttcct             gaaaaataagaaaagaatagat . . .          Exon 13                Intron 13 13F. strand CTAACAAAAGAGagggcttttcagct         acacaaattaaaagaaaaaaag . . .          Exon 14                Intron 14 14F. strand GTGGCATGCCCAGGgtggcatgcccagg         taaataaatgaatgaagtttcca . . .          Exon 15                Intron 15 15F. strand CTAAAAATTGGCaatttgtttgtttcc        tacagaaaaaacaacaaaaca . . .          Exon 16                Intron 16 16F. strand CAGTGTATCATTTGcagtgtatcatttg         gtatgttacccttcctttttcaaatt . . .16R. strand                             . . . aaagtcTAAGTGAAAA          Exon 17                Intron 17 17F. strand TTTGACAAAAGCAAtttgacaaaagcaa         gtatgttctacatatatgtgcatat . . .17R. strand                          . . . aaagagtcGGGTTATCAT          Exon 18                Intron 18 18F. strand GGCCTTTTTATAGGggcctttttatagg         taaganaagaaaatatgactcct . . .          Exon 19                Intron 19 19F. strand GAATATTATATATAgaatattatatata         gttatgtgagtgtttatatatgtgtgt . . . Notes:F: Forward strand R: Reverse strand

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1-44. (canceled)
 45. A method of detecting cancerous kidney, colon orbladder cells of a subject with cancer comprising: (A) contacting anendothelial cell of the neovasculature of the kidney, colon or bladderof the subject with an antibody or administering the antibody to thesubject, wherein the antibody binds to the extracellular domain ofprostate specific membrane antigen under conditions effective to permitbinding of the antibody to the endothelial cell so as to form a complexand the antibody has bound thereto an imaging agent, and (B) detectingthe complex, wherein detection of the complex indicates cancerouskidney, colon or bladder cells in the subject.
 46. (canceled) 47.(canceled)
 48. (canceled)
 49. (canceled)
 50. (canceled)
 52. The methodof claim 45, wherein the subject is human.
 53. The method of claim 45,wherein the imaging agent is a radioactive, a colorimetric, aluminescent, or a fluorescent agent or a standard pharmacophore.
 54. Themethod of claim 45, wherein said administering is carried outintravenously.
 55. (canceled)
 56. The method of claim 45, wherein theantibody is a monoclonal antibody or a polyclonal antibody. 57.(canceled)
 58. The method of claim 45, wherein the antibody is in acomposition further comprising a physiologically acceptable carrier,excipient, or stabilizer.
 59. The method of claim 45, wherein theantibody is in a composition further comprising a pharmaceuticallyacceptable carrier, excipient, or stabilizer.
 60. The method of claim45, wherein said contacting is carried out in a sample of urine.
 61. Themethod of claim 45, wherein said contacting is carried out in a tissuesample.